July 1, 2001CODE OF FEDERAL REGULATIONS40Parts 136 to 149Revised as of July 1, 2001Protection of EnvironmentContaining a codification of documents of general applicability and future effectAs of July 1, 2001With Ancillaries

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Table of ContentsPageExplanationvTitle 40:Chapter I—Environmental Protection Agency (Continued)3Finding Aids:Material Approved for Incorporation by Reference837Table of CFR Titles and Chapters855Alphabetical List of Agencies Appearing in the CFR873List of CFR Sections Affected883

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INCORPORATION BY REFERENCE

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What is a proper incorporation by reference? The Director of the Federal Register will approve an incorporation by reference only when the requirements of 1 CFR part 51 are met. Some of the elements on which approval is based are:

(a) The incorporation will substantially reduce the volume of material published in the Federal Register.

(b) The matter incorporated is in fact available to the extent necessary to afford fairness and uniformity in the administrative process.

(c) The incorporating document is drafted and submitted for publication in accordance with 1 CFR part 51.

Properly approved incorporations by reference in this volume are listed in the Finding Aids at the end of this volume.

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Raymond A. Mosley,Director,Office of the Federal Register.July 1, 2001.THIS TITLE

Title 40—Protection of Environment is composed of twenty-eight volumes. The parts in these volumes are arranged in the following order: parts 1-49, parts 50-51, part 52 (52.01-52.1018), part 52 (52.1019-End), parts 53-59, part 60 (60.1-End), part 60 (Appendices), parts 61-62, part 63 (63.1-63.599), part 63 (63.600-1-63.1199), part 63 (63.1200-End), parts 64-71, parts 72-80, parts 81-85, part 86 (86.1-86.599-99) part 86 (86.600-1-End), parts 87-99, parts 100-135, parts 136-149, parts 150-189, parts 190-259, parts 260-265, parts 266-299, parts 300-399, parts 400-424, parts 425-699, parts 700-789, and part 790 to End. The contents of these volumes represent all current regulations codified under this title of the CFR as of July 1, 2001.

Chapter I—Environmental Protection Agency appears in all twenty-four volumes. A Pesticide Tolerance Commodity/Chemical Index and Crop Grouping Commodities Index appear in parts 150-189. A Toxic Substances Chemical—CAS Number Index appears in parts 700-789 and part 790 to End. Redesignation Tables appear in the volumes containing parts 50-51, parts 150-189, and parts 700-789. Regulations issued by the Council on Environmental Quality appear in the volume containing part 790 to End. The OMB control numbers for title 40 appear in § 9.1 of this chapter.

The procedures prescribed herein shall, except as noted in § 136.5, be used to perform the measurements indicated whenever the waste constituent specified is required to be measured for:

(a) An application submitted to the Administrator, or to a State having an approved NPDES program for a permit under section 402 of the Clean Water Act of 1977, as amended (CWA), and/or to reports required to be submitted under NPDES permits or other requests for quantitative or qualitative effluent data under parts 122 to 125 of title 40, and,

(b) Reports required to be submitted by discharges under the NPDES established by parts 124 and 125 of this chapter, and,

(c) Certifications issued by States pursuant to section 401 of the CWA, as amended.

(a) Act means the Clean Water Act of 1977, Pub. L. 95-217, 91 Stat. 1566, et seq. (33 U.S.C. 1251 et seq.) (The Federal Water Pollution Control Act Amendments of 1972 as amended by the Clean Water Act of 1977).

(b) Administrator means the Administrator of the U.S. Environmental Protection Agency.

(c) Regional Administrator means one of the EPA Regional Administrators.

(d) Director means the Director of the State Agency authorized to carry out an approved National Pollutant Discharge Elimination System Program under section 402 of the Act.

(e) National Pollutant Discharge Elimination System (NPDES) means the national system for the issuance of permits under section 402 of the Act and includes any State or interstate program which has been approved by the Administrator, in whole or in part, pursuant to section 402 of the Act.

(f) Detection limit means the minimum concentration of an analyte (substance) that can be measured and reported with a 99% confidence that the analyte concentration is greater than zero as determined by the procedure set forth at appendix B of this part.

(a) Parameters or pollutants, for which methods are approved, are listed together with test procedure descriptions and references in Tables IA, IB, IC, ID, IE, and IF. The full text of the referenced test procedures are incorporated by reference into Tables IA, IB, IC, ID, IE, and IF. The references and the sources which are available are given in paragraph (b) of this section. These test procedures are incorporated as they exist on the day of approval and a notice of any change in these test procedures will be published in the Federal Register. The discharge parameter values for which reports are required must be determined by one of the standard analytical test procedures incorporated by reference and described in Tables IA, IB, IC, ID, IE, and IF, or by any alternate test procedure which has been approved by the Administrator under the provisions of paragraph (d) of this section and §§ 136.4 and 136.5. Under certain circumstances (paragraph (b) or (c) of this section or 40 CFR 401.13) other test procedures may be used that may be more advantageous when such other test procedures have been previously approved by the Regional Administrator of the Region in which the discharge will occur, and providing the Director of the State in which such discharge will occur does not object to the use of such alternate test procedure.

Notes to Table IA:1 The method must be specified when results are reported.2 A 0.45 um membrane filter (MF) or other pore size certified by the manufacturer to fully retain organisms to be cultivated and to be free of extractables which could interfere with their growth.3 USEPA. 1978. Microbiological Methods for Monitoring the Environment, Water, and Wastes. Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio. EPA/600/8-78/017.4 APHA. 1992. Standard Methods for the Examination of Water and Wastewater. American Public Health Association. 18th Edition. Amer. Publ. Hlth. Assoc., Washington, DC.5 USGS. 1989. U.S. Geological Survey Techniques of Water-Resources Investigations, Book 5, Laboratory Analysis, Chapter A4, Methods for Collection and Analysis of Aquatic Biological and Microbiological Samples, U.S. Geological Survey, U.S. Department of Interior, Reston, Virginia.6 Because the MF technique usually yields low and variable recovery from chlorinated wastewaters, the Most Probable Number method will be required to resolve any controversies.7 USEPA. 1993. Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms. Fourth Edition. Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio. August 1993, EPA/600/4-90/027F.8 USEPA. 1994. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Third Edition. Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency USEPA. 1994, Cincinnati, Ohio (July 1994, EPA/600/4-91/002).9 Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms. Second Edition. Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio (July 1994, EPA/600/4-91/003). These methods do not apply to marine waters of the Pacific Ocean.Table IB.—List of Approved Inorganic Test ProceduresParameter, units and methodReference (method number or page)EPA 1,35STD methods

(b) The full texts of the methods from the following references which are cited in Tables IA, IB, IC, ID, IE,and IF are incorporated by reference into this regulation and may be obtained from the sources identified. All costs cited are subject to change and must be verified from the indicated sources. The full texts of all the test procedures cited are available for inspection at the National Exposure Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, 26 West Martin Luther King Dr., Cincinnati, OH 45268 and the Office of the Federal Register, 800 North Capitol Street, NW., Suite 700, Washington, DC.

References, Sources, Costs, and Table Citations:

(1) The full texts of Methods 601-613, 624, 625, 1613, 1624, and 1625 are printed in appendix A of this part 136. The full text for determining the method detection limit when using the test procedures is given in appendix B of this part 136. The full text of Method 200.7 is printed in appendix C of this part 136. Cited in: Table IB, Note 5; Table IC, Note 2; and Table ID, Note 2.

(18) “American National Standard on Photographic Processing Effluents,” April 2, 1975. Available from: American National Standards Institute, 1430 Broadway, New York, New York 10018. Table IB, Note 9.

(19) “An Investigation of Improved Procedures for Measurement of Mill Effluent and Receiving Water Color,” NCASI Technical Bulletin No. 253, December 1971. Available from: National Council of the Paper Industry for Air and Stream Improvements, Inc., 260 Madison Avenue, New York, NY 10016. Cost available from publisher. Table IB, Note 18.

(40) EPA Methods 1666, 1667, and 1671 listed in the table above are published in the compendium titled Analytical Methods for the Determination of Pollutants in Pharmaceutical Manufacturing Industry Wastewaters (EPA 821-B-98-016). EPA Methods 502.2 and 524.2 have been incorporated by reference into 40 CFR 141.24 and are in Methods for the Determination of Organic Compounds in Drinking Water, EPA-600/4-88-039, December 1988, Revised, July 1991, and Methods for the Determination of Organic Compounds in Drinking Water-Supplement II, EPA-600/R-92-129, August 1992, respectively. These EPA test method compendia are available from the National Technical Information Service, NTIS PB91-231480 and PB92-207703, U.S. Department of Commerce, 5285 Port Royal Road, Springfield, Virginia 22161. The toll-free number is 800-553-6847. ASTM test methods D3371, D3695, and D4763 are available from the American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959.

Editorial Note:

At 64 FR 30434, June 8, 1999, the following paragraph (40) was added, effective July 8, 1999; however paragraph (40) was previously added to the 1999 volume.

(41) USEPA, January 1999 Errata for the Effluent and Receiving Water Testing Manuals: Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms; Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms; and Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms. U.S. Environmental Protection Agency, Office of Research and Development, Duluth, MN. EPA-600/R-98/182.

(c) Under certain circumstances the Regional Administrator or the Director in the Region or State where the discharge will occur may determine for a particular discharge that additional parameters or pollutants must be reported. Under such circumstances, additional test procedures for analysis of pollutants may be specified by the Regional Administrator, or the Director upon the recommendation of the Director of the Environmental Monitoring Systems Laboratory—Cincinnati.

(e) Sample preservation procedures, container materials, and maximum allowable holding times for parameters cited in Tables IA, IB, IC, ID, and IE are prescribed in Table II. Any person may apply for a variance from the prescribed preservation techniques, container materials, and maximum holding times applicable to samples taken from a specific discharge. Applications for variances may be made by letters to the Regional Administrator in the Region in which the discharge will occur. Sufficient data should be provided to assure such variance does not adversely affect the integrity of the sample. Such data will be forwarded, by the Regional Administrator, to the Director of the Environmental Monitoring Systems Laboratory—Cincinnati, Ohio for technical review and recommendations for action on the variance application. Upon receipt of the recommendations from the Director of the Environmental Monitoring Systems Laboratory, the Regional Administrator may grant a variance applicable to the specific charge to the applicant. A decision to approve or deny a variance will be made within 90 days of receipt of the application by the Regional Administrator.

Table II—Required Containers, Preservation Techniques, and Holding TimesParameter No./nameContainer 1Preservation 2,3Maximum holding time 4Table IA—Bacteria Tests:1-4 Coliform, fecal and totalP,GCool, 4C, 0.008% Na2S2O356 hours.5 Fecal streptococciP,GCool, 4C, 0.008% Na2S2O356 hours.Table IA—Aquatic Toxicity Tests:6-10 Toxicity, acute and chronicP,GCool, 4 °C 1636 hours.Table IB—Inorganic Tests:1. AcidityP, GCool, 4°C14 days.2. AlkalinityP, G......doDo.4. AmmoniaP, GCool, 4°C, H2SO4 to pH<228 days.9. Biochemical oxygen demandP, GCool, 4°C48 hours.10. BoronP, PFTE, or QuartzHNO3 TO pH<26 months.11. BromideP, GNone required28 days.14. Biochemical oxygen demand, carbonaceousP, GCool, 4°C48 hours.15. Chemical oxygen demandP, GCool, 4°C, H2SO4 to pH<228 days.16. ChlorideP, GNone requiredDo.17. Chlorine, total residualP, G......doAnalyze immediately.21. ColorP, GCool, 4°C48 hours.23-24. Cyanide, total and amenable to chlorinationP, GCool, 4°C, NaOH to pH>12, 0.6g ascorbic acid 514 days.625. FluoridePNone required28 days.27. HardnessP, GHNO3 to pH<2, H2SO4 to pH<26 months.28. Hydrogen ion (pH)P, GNone requiredAnalyze immediately.31, 43. Kjeldahl and organic nitrogenP, GCool, 4°C, H2SO4 to pH<228 days.Metals:718. Chromium VIP, GCool, 4°C24 hours.35. MercuryP, GHNO3 to pH<228 days.3, 5-8, 12, 13, 19, 20, 22, 26, 29, 30, 32-34, 36, 37, 45, 47, 51, 52, 58-60, 62, 63, 70-72, 74, 75. Metals, except boron, chromium VI and mercuryP, G......do6 months.38. NitrateP, GCool, 4°C48 hours.39. Nitrate-nitriteP, GCool, 4°C, H2SO4 to pH<228 days.40. NitriteP, GCool, 4°C48 hours.41. Oil and greaseGCool to 4°C, HCl or H2SO4 to pH<228 days.42. Organic CarbonP, GCool to 4 °C HC1 or H2SO4 or H3PO4, to pH<228 days.44. OrthophosphateP, GFilter immediately, Cool, 4°C48 hours.46. Oxygen, Dissolved ProbeG Bottle and topNone requiredAnalyze immediately.47. Winkler......doFix on site and store in dark8 hours.48. PhenolsG onlyCool, 4°C, H2SO4 to pH<228 days.49. Phosphorus (elemental)GCool, 4°C48 hours.50. Phosphorus, totalP, GCool, 4°C, H2SO4 to pH<228 days.53. Residue, totalP, GCool, 4°C7 days.54. Residue, FilterableP, G......do7 days.55. Residue, Nonfilterable (TSS)P, G......do7 days.56. Residue, SettleableP, G......do48 hours.57. Residue, volatileP, G......do7 days.61. SilicaP, PFTE, or QuartzCool, 4 °C28 days.64. Specific conductanceP, G......doDo.65. SulfateP, G......doDo.66. SulfideP, GCool, 4°C add zinc acetate plus sodium hydroxide to pH>97 days.67. SulfiteP, GNone requiredAnalyze immediately.68. SurfactantsP ,GCool, 4°C48 hours.69. TemperatureP, GNone requiredAnalyze.73. TurbidityP, GCool, 4°C48 hours.Table IC—Organic Tests 813, 18-20, 22, 24-28, 34-37, 39-43, 45-47, 56, 76, 104, 105, 108-111, 113. Purgeable HalocarbonsG, Teflon-lined septumCool, 4 °C, 0.008% Na2S2O35.14 days.6, 57, 106. Purgeable aromatic hydrocarbons......doCool, 4 °C, 0.008% Na2S2O3,5 HCl to pH29Do.3, 4. Acrolein and acrylonitrile......doCool, 4 °C, 0.008% Na2S2O3,5 adjust pH to 4-510Do.23, 30, 44, 49, 53, 77, 80, 81, 98, 100, 112. Phenols 11G, Teflon-lined cap.Cool, 4 °C, 0.008% Na2S2O357 days until extraction; 40 days after extraction.7, 38. Benzidines 11......do......do7 days until extraction.1314, 17, 48, 50-52. Phthalate esters 11......doCool, 4 °C7 days until extraction; 40 days after extraction.82-84. Nitrosamines 1114......doCool, 4 °C, 0.008% Na2S2O3,5 store in darkDo.88-94. PCBs 11.....doCool, 4 °CDo.54, 55, 75, 79. Nitroaromatics and isophorone 11......doCool, 4 °C, 0.008% Na2S2O3,5 store in darkDo.1, 2, 5, 8-12, 32, 33, 58, 59, 74, 78, 99, 101. Polynuclear aromatic hydrocarbons 11......do......doDo.15, 16, 21, 31, 87. Haloethers 11......doCool, 4 °C, 0.008% Na2S2O35Do.29, 35-37, 63-65, 73, 107. Chlorinated hydrocarbons 11......doCool, 4 °CDo.60-62, 66-72, 85, 86, 95-97, 102, 103. CDDs/CDFs 11aqueous: field and lab preservation.GCool, 0-4 °C, pH<9, 0.008% Na2S2O351 year.Solids, mixed phase, and tissue: field preservation.......doCool, <4 °C7 days.Solids, mixed phase, and tissue: lab preservation......doFreeze, <−10 °C1 year.Table ID—Pesticides Tests:1-70. Pesticides 11......doCool, 4°C, pH 5-9 15Do.Table IE—Radiological Tests:1-5. Alpha, beta and radiumP, GHNO3 to pH<26 months.Table II Notes1 Polyethylene (P) or glass (G). For microbiology, plastic sample containers must be made of sterilizable materials (polypropylene or other autoclavable plastic).2 Sample preservation should be performed immediately upon sample collection. For composite chemical samples each aliquot should be preserved at the time of collection. When use of an automated sampler makes it impossible to preserve each aliquot, then chemical samples may be preserved by maintaining at 4°C until compositing and sample splitting is completed.3 When any sample is to be shipped by common carrier or sent through the United States Mails, it must comply with the Department of Transportation Hazardous Materials Regulations (49 CFR part 172). The person offering such material for transportation is responsible for ensuring such compliance. For the preservation requirements of Table II, the Office of Hazardous Materials, Materials Transportation Bureau, Department of Transportation has determined that the Hazardous Materials Regulations do not apply to the following materials: Hydrochloric acid (HCl) in water solutions at concentrations of 0.04% by weight or less (pH about 1.96 or greater); Nitric acid (HNO3) in water solutions at concentrations of 0.15% by weight or less (pH about 1.62 or greater); Sulfuric acid (H2SO4) in water solutions at concentrations of 0.35% by weight or less (pH about 1.15 or greater); and Sodium hydroxide (NaOH) in water solutions at concentrations of 0.080% by weight or less (pH about 12.30 or less).4 Samples should be analyzed as soon as possible after collection. The times listed are the maximum times that samples may be held before analysis and still be considered valid. Samples may be held for longer periods only if the permittee, or monitoring laboratory, has data on file to show that for the specific types of samples under study, the analytes are stable for the longer time, and has received a variance from the Regional Administrator under § 136.3(e). Some samples may not be stable for the maximum time period given in the table. A permittee, or monitoring laboratory, is obligated to hold the sample for a shorter time if knowledge exists to show that this is necessary to maintain sample stability. See § 136.3(e) for details. The term “analyze immediately” usually means within 15 minutes or less of sample collection.5 Should only be used in the presence of residual chlorine.6 Maximum holding time is 24 hours when sulfide is present. Optionally all samples may be tested with lead acetate paper before pH adjustments in order to determine if sulfide is present. If sulfide is present, it can be removed by the addition of cadmium nitrate powder until a negative spot test is obtained. The sample is filtered and then NaOH is added to pH 12.7 Samples should be filtered immediately on-site before adding preservative for dissolved metals.8 Guidance applies to samples to be analyzed by GC, LC, or GC/MS for specific compounds.9 Sample receiving no pH adjustment must be analyzed within seven days of sampling.10 The pH adjustment is not required if acrolein will not be measured. Samples for acrolein receiving no pH adjustment must be analyzed within 3 days of sampling.11 When the extractable analytes of concern fall within a single chemical category, the specified preservative and maximum holding times should be observed for optimum safeguard of sample integrity. When the analytes of concern fall within two or more chemical categories, the sample may be preserved by cooling to 4°C, reducing residual chlorine with 0.008% sodium thiosulfate, storing in the dark, and adjusting the pH to 6-9; samples preserved in this manner may be held for seven days before extraction and for forty days after extraction. Exceptions to this optional preservation and holding time procedure are noted in footnote 5 (re the requirement for thiosulfate reduction of residual chlorine), and footnotes 12, 13 (re the analysis of benzidine).12 If 1,2-diphenylhydrazine is likely to be present, adjust the pH of the sample to 4.0±0.2 to prevent rearrangement to benzidine.13 Extracts may be stored up to 7 days before analysis if storage is conducted under an inert (oxidant-free) atmosphere.14 For the analysis of diphenylnitrosamine, add 0.008% Na2S2O3 and adjust pH to 7-10 with NaOH within 24 hours of sampling.15 The pH adjustment may be performed upon receipt at the laboratory and may be omitted if the samples are extracted within 72 hours of collection. For the analysis of aldrin, add 0.008% Na2S2O3.16 Sufficient ice should be placed with the samples in the shipping container to ensure that ice is still present when the samples arrive at the laboratory. However, even if ice is present when the samples arrive, it is necessary to immediately measure the temperature of the samples and confirm that the 4C temperature maximum has not been exceeded. In the isolated cases where it can be documented that this holding temperature can not be met, the permittee can be given the option of on-site testing or can request a variance. The request for a variance should include supportive data which show that the toxicity of the effluent samples is not reduced because of the increased holding temperature. [38 FR 28758, Oct. 16, 1973, as amended at 41 FR 52781, Dec. 1, 1976; 49 FR 43251, 43258, 43259, Oct. 26, 1984; 50 FR 691, 692, 695, Jan. 4, 1985; 51 FR 23693, June 30, 1986; 52 FR 33543, Sept. 3, 1987; 55 FR 24534, June 15, 1990; 55 FR 33440, Aug. 15, 1990; 56 FR 50759, Oct. 8, 1991; 57 FR 41833, Sept. 11, 1992; 58 FR 4505, Jan. 31, 1994; 60 FR 17160, Apr. 4, 1995; 60 FR 39588, 39590, Aug. 2, 1995; 60 FR 44672, Aug. 28, 1995; 60 FR 53542, 53543, Oct. 16, 1995; 62 FR 48403, 48404, Sept. 15, 1997; 63 FR 50423, Sept. 21, 1998; 64 FR 4978, Feb. 2, 1999; 64 FR 10392, Mar. 4, 1999; 64 FR 26327, May 14, 1999; 64 FR 30433, 30434, June 8, 1999; 64 FR 73423, Dec. 30, 1999]Effective Date Note:

At 66 FR 32776, June 18, 2001, § 136.3 was amended by redesignating paragraph (b)(41) as (b)(42) and by redesignating the second paragraph (b)(40) as new (b)(41) and revising it, effective July 18, 2001. For the convenience of the user, the revised text is set forth as follows:

(a) Any person may apply to the Regional Administrator in the Region where the discharge occurs for approval of an alternative test procedure.

(b) When the discharge for which an alternative test procedure is proposed occurs within a State having a permit program approved pursuant to section 402 of the Act, the applicant shall submit his application to the Regional Administrator through the Director of the State agency having responsibility for issuance of NPDES permits within such State.

(c) Unless and until printed application forms are made available, an application for an alternate test procedure may be made by letter in triplicate. Any application for an alternate test procedure under this paragraph (c) shall:

(1) Provide the name and address of the responsible person or firm making the discharge (if not the applicant) and the applicable ID number of the existing or pending permit, issuing agency, and type of permit for which the alternate test procedure is requested, and the discharge serial number.

(2) Identify the pollutant or parameter for which approval of an alternate testing procedure is being requested.

(3) Provide justification for using testing procedures other than those specified in Table I.

(4) Provide a detailed description of the proposed alternate test procedure, together with references to published studies of the applicability of the alternate test procedure to the effluents in question.

(d) An application for approval of an alternate test procedure for nationwide use may be made by letter in triplicate to the Director, Analytical Methods Staff, Office of Science and Technology (4303), Office of Water, U.S. Environmental Protection Agency, 1200 Pennsylvania Ave., NW., Washington, DC 20460. Any application for an alternate test procedure under this paragraph (d) shall:

(1) Provide the name and address of the responsible person or firm making the application.

(2) Identify the pollutant(s) or parameter(s) for which nationwide approval of an alternate testing procedure is being requested.

(3) Provide a detailed description of the proposed alternate procedure, together with references to published or other studies confirming the general applicability of the alternate test procedure to the pollutant(s) or para-meter(s) in waste water discharges from representative and specified industrial or other categories.

(4) Provide comparability data for the performance of the proposed alternate test procedure compared to the performance of the approved test procedures.

(a) The Regional Administrator of the region in which the discharge will occur has final responsibility for approval of any alternate test procedure proposed by the responsible person or firm making the discharge.

(b) Within thirty days of receipt of an application, the Director will forward such application proposed by the responsible person or firm making the discharge, together with his recommendations, to the Regional Administrator. Where the Director recommends rejection of the application for scientific and technical reasons which he provides, the Regional Administrator shall deny the application, and shall forward a copy of the rejected application and his decision to the Director of the State Permit Program and to the Director of the Analytical Methods Staff, Washington, DC.

(c) Before approving any application for an alternate test procedure proposed by the responsible person or firm making the discharge, the Regional Administrator shall forward a copy of the application to the Director of the Analytical Methods Staff, Washington, DC.

(d) Within ninety days of receipt by the Regional Administrator of an application for an alternate test procedure, proposed by the responsible person or firm making the discharge, the Regional Administrator shall notify the applicant and the appropriate State agency of approval or rejection, or shall specify the additional information which is required to determine whether to approve the proposed test procedure. Prior to the expiration of such ninety day period, a recommendation providing the scientific and other technical basis for acceptance or rejection will be forwarded to the Regional Administrator by the Director of the Analytical Methods Staff, Washington, DC. A copy of all approval and rejection notifications will be forwarded to the Director, Analytical Methods Staff, Washington, DC, for the purposes of national coordination.

(e) Approval for nationwide use. (1) Within sixty days of the receipt by the Director of the Analytical Methods Staff, Washington, DC, of an application for an alternate test procedure for nationwide use, the Director of the Analytical Methods Staff shall notify the applicant in writing whether the application is complete. If the application is incomplete, the applicant shall be informed of the information necessary to make the application complete.

(2) Within ninety days of the receipt of a complete package, the Analytical Methods Staff shall perform any analysis necessary to determine whether the alternate method satisfies the applicable requirements of this part, and the Director of the Analytical Methods Staff shall recommend to the Administrator that he/she approve or reject the application and shall also notify the applicant of such recommendation.

(3) As expeditiously as practicable, an alternate method determined by the Administrator to satisfy the applicable requirements of this part shall be proposed by EPA for incorporation in subsection 136.3 of 40 CFR part 136. EPA shall make available for review all the factual bases for its proposal, including any performance data submitted by the applicant and any available EPA analysis of those data.

(4) Following a period of public comment, EPA shall, as expeditiously as practicable, publish in the Federal Register a final decision to approve or reject the alternate method.

1.2This is a purge and trap gas chro-ma-tographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 624 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for most of the parameters listed above.

1.3The method detection limit (MDL, defined in Section 12.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5This method is restricted to use by or under the supervision of analysts experienced in the operation of a purge and trap system and a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1An inert gas is bubbled through a 5-mL water sample contained in a specially-designed purging chamber at ambient temperature. The halocarbons are efficiently transferred from the aqueous phase to the vapor phase. The vapor is swept through a sorbent trap where the halocarbons are trapped. After purging is completed, the trap is heated and backflushed with the inert gas to desorb the halocarbons onto a gas chromatographic column. The gas chromatograph is temperature programmed to separate the halocarbons which are then detected with a halide-specific detector.2,3

2.2The method provides an optional gas chromatographic column that may be helpful in resolving the compounds of interest from interferences that may occur.

3. Interferences

3.1Impurities in the purge gas and organic compounds outgassing from the plumbing ahead of the trap account for the majority of contamination problems. The analytical system must be demonstrated to be free from contamination under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3. The use of non-Teflon plastic tubing, non-Teflon thread sealants, or flow controllers with rubber components in the purge and trap system should be avoided.

3.2Samples can be contaminated by diffusion of volatile organics (particularly fluorocarbons and methylene chloride) through the septum seal ilto the sample during shipment and storage. A field reagent blank prepared from reagent water and carried through the sampling and handling protocol can serve as a check on such contamination.

3.3Contamination by carry-over can occur whenever high level and low level samples are sequentially analyzed. To reduce carry-over, the purging device and sample syringe must be rinsed with reagent water between sample analyses. Whenever an unusually concentrated sample is encountered, it should be followed by an analysis of reagent water to check for cross contamination. For samples containing large amounts of water-soluble materials, suspended solids, high boiling compounds or high organohalide levels, it may be necessary to wash out the purging device with a detergent solution, rinse it with distilled water, and then dry it in a 105°C oven between analyses. The trap and other parts of the system are also subject to contamination; therefore, frequent bakeout and purging of the entire system may be required.

4. Safety

4.1The toxicity or carcinogenicity of each -reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 4-6 for the information of the analyst.

4.2The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: carbon tetrachloride, chloroform, 1,4-dichlorobenzene, and vinyl chloride. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials

5.1Sampling equipment, for discrete sampling.

5.1.1Vial—25-mL capacity or larger, equipped with a screw cap with a hole in the center (Pierce #13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 °C before use.

5.2Purge and trap system—The purge and trap system consists of three separate pieces of equipment: a purging device, trap, and desorber. Several complete systems are now commercially available.

5.2.1The purging device must be designed to accept 5-mL samples with a water column at least 3 cm deep. The gaseous head space between the water column and the trap must have a total volume of less than 15 mL. The purge gas must pass through the water column as finely divided bubbles with a diameter of less than 3 mm at the origin. The purge gas must be introduced no more than 5 mm from the base of the water column. The purging device illustrated in Figure 1 meets these design criteria.

5.2.2The trap must be at least 25 cm long and have an inside diameter of at least 0.105 in. The trap must be packed to contain the following minimum lengths of adsorbents: 1.0 cm of methyl silicone coated packing (Section 6.3.3), 7.7 cm of 2,6-diphenylene oxide polymer (Section 6.3.2), 7.7 cm of silica gel (Section 6.3.4), 7.7 cm of coconut charcoal (Section 6.3.1). If it is not necessary to analyze for dichlorodifluoromethane, the charcoal can be eliminated, and the polymer section lengthened to 15 cm. The minimum specifications for the trap are illustrated in Figure 2.

5.2.3The desorber must be capable of rapidly heating the trap to 180 °C. The polymer section of the trap should not be heated higher than 180 °C and the remaining sections should not exceed 200 °C. The desorber illustrated in Figure 2 meets these design criteria.

5.2.4The purge and trap system may be assembled as a separate unit or be coupled to a gas chromatograph as illustrated in Figures 3 and 4.

5.3Gas chromatograph—An analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.3.1Column 1—8 ft long x 0.1 in. ID stainless steel or glass, packed with 1% SP-1000 on Carbopack B (60/80 mesh) or equivalent. This column was used to develop the method performance statements in Section 12. Guidelines for the use of alternate column packings are provided in Section 10.1.

5.3.3Detector—Electrolytic conductivity or microcoulometric detector. These types of detectors have proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1). The electrolytic conductivity detector was used to develop the method performance statements in Section 12. Guidelines for the use of alternate detectors are provided in Section 10.1.

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.1.1Reagent water can be generated by passing tap water through a carbon filter bed containing about 1 lb of activated carbon (Filtrasorb-300, Calgon Corp., or equivalent).

6.1.2A water purification system (Millipore Super-Q or equivalent) may be used to generate reagent water.

6.1.3Reagent water may also be prepared by boiling water for 15 min. Subsequently, while maintaining the temperature at 90°C, bubble a contaminant-free inert gas through the water for 1 h. While still hot, transfer the water to a narrow mouth screw-cap bottle and seal with a Teflon-lined septum and cap.

6.5Stock standard solutions—Stock standard solutions may be prepared from pure standard materials or purchased as certified solutions. Prepare stock standard solutions in methanol using assayed liquids or gases as appropriate. Because of the toxicity of some of the organohalides, primary dilutions of these materials should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be used when the analyst handles high concentrations of such materials.

6.5.1Place about 9.8 mL of methanol into a 10-mL ground glass stoppered volumetric flask. Allow the flask to stand, unstoppered, for about 10 min or until all alcohol wetted surfaces have dried. Weigh the flask to the learest 0.1 mg.

6.5.2Add the assayed reference material:

6.5.2.1Liquid—Using a 100 μL syringe, immediately add two or more drops of assayed reference material to the flask, then reweigh. Be sure that the drops fall directly into the alcohol without contacting the neck of the flask.

6.5.2.2Gases—To prepare standards for any of the six halocarbons that boil below 30 ° C (bromomethane, chloroethane, chloro-methane, dichlorodifluoromethane, -trichlorofluoromethane, vinyl chloride), -fill a 5-mL valved gas-tight syringe with the reference standard to the 5.0-mL mark. Lower the needle to 5 mm above the methanol meniscus. Slowly introduce the reference standard above the surface of the liquid (the heavy gas will rapidly dissolve into the methanol).

6.5.3Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in μg/μL from the net gain in weight. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the malufacturer or by an independent source.

6.5.4Transfer the stock standard solution into a Teflon-sealed screw-cap bottle. Store, with minimal headspace, at −10 to −20 °C and protect from light.

6.5.5Prepare fresh standards weekly for the six gases and 2-chloroethylvinyl ether. All other standards must be replaced after one month, or sooner if comparison with check standards indicates a problem.

6.6Secondary dilution standards—Using stock standard solutions, prepare secondary dilution standards in methanol that contain the compounds of interest, either singly or mixed together. The secondary dilution standards should be prepared at concentrations such that the aqueous calibration standards prepared in Section 7.3.1 or 7.4.1 will bracket the working range of the analytical system. Secondary dilution standards should be stored with minimal headspace and should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1Assemble a purge and trap system that meets the specifications in Section 5.2. Condition the trap overnight at 180 °C by backflushing with an inert gas flow of at least 20 mL/min. Condition the trap for 10 min once daily prior to use.

7.2Connect the purge and trap system to a gas chromatograph. The gas chromatograph must be operated using temperature and flow rate conditions equivalent to those given in Table 1. Calibrate the purge and trap-gas chromatographic system using either the external standard technique (Section 7.3) or the internal standard technique (Section 7.4).

7.3External standard calibration procedure:

7.3.1Prepare calibration standards at a miminum of three concentration levels for each parameter by carefully adding 20.0 μL of one or more secondary dilution standards to 100, 500, or 1000 μL of reagent water. A 25-μL syringe with a 0.006 in. ID needle should be used for this operation. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector. These aqueous standards can be stored up to 24 h, if held in sealed vials with zero headspace as described in Section 9.2. If not so stored, they must be discarded after 1 h.

7.3.2Analyze each calibration standard according to Section 10, and tabulate peak height or area responses versus the concentration in the standard. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to concentration (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.4Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples. The compounds recommended for use as surrogate spikes in Section 8.7 have been used successfully as internal standards, because of their generally unique retention times.

7.4.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest as described in Section 7.3.1.

7.4.2Prepare a spiking solution containing each of the internal standards using the procedures described in Sections 6.5 and 6.6. It is recommended that the secondary dilution standard be prepared at a concentration of 15 μg/mL of each internal standard compound. The addition of 10 μL of this standard to 5.0 mL of sample or calibration standard would be equivalent to 30 μg/L.

7.4.3Analyze each calibration standard according to Section 10, adding 10 μL of internal standard spiking solution directly to the syringe (Section 10.4). Tabulate peak height or area responses against concentration for each compound and internal standard, and calculate response factors (RF) for each compound using Equation 1.

EC15NO91.094Equation 1where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard.Cs=Concentration of the parameter to be measured.If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.5The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of a QC check sample.

7.5.1Prepare the QC check sample as described in Section 8.2.2.

7.5.2Analyze the QC check sample according to Section 10.

7.5.3For each parameter, compare the response (Q) with the corresponding calibration acceptance criteria found in Table 2. If the responses for all parameters of interest fall within the designated ranges, analysis of actual samples can begin. If any individual Q falls outside the range, proceed according to Section 7.5.4.

Note:

The large number of parameters in Table 2 present a substantial probability that one or more will not meet the calibration acceptance criteria when all parameters are analyzed.

7.5.4Repeat the test only for those parameters that failed to meet the calibration acceptance criteria. If the response for a parameter does not fall within the range in this second test, a new calibration curve, calibration factor, or RF must be prepared for that parameter according to Section 7.3 or 7.4.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Section 10.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Each day, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system are under control.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 10 μg/mL in methanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.3Analyze four 5-mL aliquots of the well-mixed QC check sample according to Section 10.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for each parameter of interest using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, then the system performance is unacceptable for that parameter.

Note:

The large number of parameters in Table 2 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.3.

8.2.6.2Beginning with Section 8.2.3, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.3.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 20 μg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.2Analyze one 5-mL sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second 5-mL sample aliquot with 10 μL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than 20 μg/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.7

8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory. If the entire list of parameters in Table 2 must be measured in the sample in Section 8.3, the probability that the analysis of a QC check standard will be required is high. In this case the QC check standard should be routinely analyzed with the spiked sample.

8.4.1Prepare the QC check standard by adding 10 μL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 5 mL of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P−2sp to P+2sp. If p=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

8.7The analyst should monitor both the performance of the analytical system and the effectiveness of the method in deal-ing with each sample matrix by spiking -each sample, standard, and reagent -water blank with surrogate halocarbons. -A combination of bromochloromethane, -2-bromo-1-chloropropane, and 1,4-dichlorobu-tane is recommended to encompass the range of the temperature program used in this method. From stock standard solutions prepared as in Section 6.5, add a volume to give 750 μg of each surrogate to 45 mL of reagent water contained in a 50-mL volumetric flask, mix and dilute to volume for a concentration of 15 ng/μL. Add 10 μL of this surrogate spiking solution directly into the 5-mL syringe with every sample and reference standard analyzed. Prepare a fresh surrogate spiking solution on a weekly basis. If the internal standard calibration procedure is being used, the surrogate compounds may be added directly to the internal standard spiking solution (Section 7.4.2).

9. Sample Collection, Preservation, and Handling

9.1All samples must be iced or refrigerated from the time of collection until analysis. If the sample contains free or combined chlorine, add sodium thiosulfate preservative (10 mg/40 mL is sufficient for up to 5 ppm Cl2) to the empty sample bottle just prior to shipping to the sampling site. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.8 Field test kits are available for this purpose.

9.2Grab samples must be collected in glass containers having a total volume of at least 25 mL. Fill the sample bottle just to overflowing in such a manner that no air bubbles pass through the sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. If preservative has been added, shake vigorously for 1 min. Maintain the hermetic seal on the sample bottle until time of analysis.

9.3All samples must be analyzed within 14 days of collection.3

10. Procedure

10.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are estimated retention times and MDL that can be achieved under these conditions. An example of the separations achieved by Column 1 is shown in Figure 5. Other packed columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

10.2Calibrate the system daily as described in Section 7.

10.3Adjust the purge gas (nitrogen or helium) flow rate to 40 mL/min. Attach the trap inlet to the purging device, and set the purge and trap system to purge (Figure 3). Open the syringe valve located on the purging device sample introduction needle.

10.4Allow the sample to come to ambient temperature prior to introducing it to the syringe. Remove the plunger from a 5-mL syringe and attach a closed syringe valve. Open the sample bottle (or standard) and carefully pour the sample into the syringe barrel to just short of overflowing. Replace the syringe plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Since this process of taking an aliquot destroys the validity of the sample for future analysis, the analyst should fill a second syringe at this time to protect against possible loss of data. Add 10.0 μL of the surrogate spiking solution (Section 8.7) and 10.0 μL of the internal standard spiking solution (Section 7.4.2), if applicable, through the valve bore, then close the valve.

10.5Attach the syringe-syringe valve assembly to the syringe valve on the purging device. Open the syringe valves and inject the sample into the purging chamber.

10.6Close both valves and purge the sample for 11.0±0.1 min at ambient temperature.

10.7After the 11-min purge time, attach the trap to the chromatograph, adjust the purge and trap system to the desorb mode (Figure 4), and begin to temperature program the gas chromatograph. Introduce the trapped materials to the GC column by rapidly heating the trap to 180 °C while backflushing the trap with an inert gas between 20 and 60 mL/min for 4 min. If rapid heating of the trap cannot be achieved, the GC column must be used as a secondary trap by cooling it to -30 °C (subambient temperature, if poor peak geometry or random retention time problems persist) instead of the initial program temperature of 45 °C

10.8While the trap is being desorbed into the gas chromatograph, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL flushes of reagent water.

10.9After desorbing the sample for 4 min, recondition the trap by returning the purge and trap system to the purge mode. Wait 15 s then close the syringe valve on the purging device to begin gas flow through the trap. The trap temperature should be maintained at -180 °C After approximately 7 min, turn off the trap heater and open the syringe valve to stop the gas flow through the trap. When the trap is cool, the next sample can be analyzed.

10.10Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

10.11If the response for a peak exceeds the working range of the system, prepare a dilution of the sample with reagent water from the aliquot in the second syringe and reanalyze.

11. Calculations

11.1Determine the concentration of individual compounds in the sample.

11.1.1If the external standard calibration procedure is used, calculate the concentration of the parameter being measured from the peak response using the calibration curve or calibration factor determined in Section 7.3.2.

11.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.4.3 and Equation 2.

Equation 2EC15NO91.095where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard.

11.2Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

12. Method Performance

12.1 The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and re-ported with 99% confidence that the value is -above zero. 1 The MDL concentration listed -in Table 1 were obtained using reagent -water.11. Similar results were achieved using -representative wastewaters. The MDL actu-ally achieved in a given analysis will -vary depending on instrument sensitivity and matrix effects.

12.2This method is recommended for use in the concentration range from the MDL to 1000×MDL. Direct aqueous injection techniques should be used to measure concentration levels above 1000×MDL.

12.3This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 8.0 to 500 μg/L.9 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

References

1. 40 CFR part 136, appendix B.

2. Bellar, T.A., and Lichtenberg, J.J. “Determining Volatile Organics at Microgram-per-Litre-Levels by Gas Chromatography,” Journal of the American Water Works Association, 66, 739 (1974).

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

Table 1—Chromatographic Conditions and Method Detection LimitsParameterRetention time (min)Column 1Column 2Method detection limit (μg/L)Chloromethane1.505.280.08Bromomethane2.177.051.18Dichlorodifluoromethane2.62nd1.81Vinyl chloride2.675.280.18Chloroethane3.338.680.52Methylene chloride5.2510.10.25Trichlorofluoromethane7.18ndnd1,1-Dichloroethene7.937.720.131,1-Dichloroethane9.3012.60.07trans-1,2-Dichloroethene10.19.380.10Chloroform10.712.10.051,2-Dichloroethane11.415.40.031,1,1-Trichloroethane12.613.10.03Carbon tetrachloride13.014.40.12Bromodichloromethane13.714.60.101,2-Dichloropropane14.916.60.04cis-1,3-Dichloropropene15.216.60.34Trichloroethene15.813.10.12Dibromochloromethane16.516.60.091,1,2-Trichloroethane16.518.10.02trans-1,3-Dichloropropene16.518.00.202-Chloroethylvinyl ether18.0nd0.13Bromoform19.219.20.201,1,2,2-Tetrachloroethane21.6nd0.03Tetrachloroethene21.715.00.03Chlorobenzene24.218.80.251,3-Dichlorobenzene34.022.40.321,2-Dichlorobenzene34.923.50.151,4-Dichlorobenzene35.422.30.24Column 1 conditions: Carbopack B (60/80 mesh) coated with 1% SP-1000 packed in an 8 ft x 0.1 in. ID stainless steel or glass column with helium carrier gas at 40 mL/min flow rate. Column temperature held at 45 °C for 3 min then programmed at 8 °C/min to 220 °C and held for 15 min.Column 2 conditions: Porisil-C (100/120 mesh) coated with n-octane packed in a 6 ft x 0.1 in. ID stainless steel or glass column with helium carrier gas at 40 mL/min flow rate. Column temperature held at 50 °C for 3 min then programmed at 6 °C/min to 170 °C and held for 4 min.nd=not determined.Table 2—Calibration and QC Acceptance Criteria—Method 601 aParameterRange for Q (μg/L)Limit for s (μg/L)Range for X (μg/L)Range P, Ps (%)Bromodichloromethane15.2-24.84.310.7-32.042-172Bromoform14.7-25.34.75.0-29.313-159Bromomethane11.7-28.37.63.4-24.5D-144Carbon tetrachloride13.7-26.35.611.8-25.343-143Chlorobenzene14.4-25.65.010.2-27.438-150Chloroethane15.4-24.64.411.3-25.246-1372-Chloroethylvinyl ether12.0-28.08.34.5-35.514-186Chloroform15.0-25.04.512.4-24.049-133Chloromethane11.9-28.17.4D-34.9D-193Dibromochloromethane13.1-26.96.37.9-35.124-1911,2-Dichlorobenzene14.0-26.05.51.7-38.9D-2081,3-Dichlorobenzene9.9-30.19.16.2-32.67-1871,4-Dichlorobenzene13.9-26.15.511.5-25.542-1431,1-Dichloroethane16.8-23.23.211.2-24.647-1321,2-Dichloroethane14.3-25.75.213.0-26.551-1471,1-Dichloroethene12.6-27.46.610.2-27.328-167trans-1,2-Dichloroethene12.8-27.26.411.4-27.138-1551,2-Dichloropropane14.8-25.25.210.1-29.944-156cis-1,3-Dichloropropene12.8-27.27.36.2-33.822-178trans-1,3-Dichloropropene12.8-27.27.36.2-33.822-178Methylene chloride15.5-24.54.07.0-27.625-1621,1,2,2-Tetrachloroethane9.8-30.29.26.6-31.88-184Tetrachloroethene14.0-26.05.48.1-29.626-1621,1,1-Trichloroethane14.2-25.84.910.8-24.841-1381,1,2-Trichloroethane15.7-24.33.99.6-25.439-136Trichloroethene15.4-24.64.29.2-26.635-146Trichlorofluoromethane13.3-26.76.07.4-28.121-156Vinyl chloride13.7-26.35.78.2-29.928-163a Criteria were calculated assuming a QC check sample concentration of 20 μg/L.Q=Concentration measured in QC check sample, in μg/L (Section 7.5.3).s=Standard deviation of four recovery measurements, in μg/L (Section 8.2.4).X=Average recovery for four recovery measurements, in μg/L (Section 8.2.4).P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).D=Detected; result must be greater than zero.Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3. Table 3.—Method Accuracy and Precision as Functions of Concentration—Method 601ParameterAccuracy, as recovery, X′ (μg/L)Single analyst precision, sr′ (μg/L)Overall precision, S′ (μg/L)Bromodichloromethane1.12C−1.020.11X+0.040.20X+1.00Bromoform0.96C−2.050.12X+0.580.21X+2.41Bromomethane0.76C−1.270.28X+0.270.36X+0.94Carbon tetrachloride0.98C−1.040.15X+0.380.20X+0.39Chlorobenzene1.00C−1.230.15X−0.020.18X+1.21Choroethane0.99C−1.530.14X−0.130.17X+0.632-Chloroethylvinyl ether a1.00C0.20X0.35XChloroform0.93C−0.390.13X+0.150.19X−0.02Chloromethane0.77C+0.180.28X−0.310.52X+1.31Dibromochloromethane0.94C+2.720.11X+1.100.24X+1.681,2-Dichlorobenzene0.93C+1.700.20X+0.970.13X+6.131,3-Dichlorobenzene0.95C+0.430.14X+2.330.26X+2.341,4-Dichlorobenzene0.93C−0.090.15X+0.290.20X+0.411,1-Dichloroethane0.95C−1.080.09X+0.170.14X+0.941,2-Dichloroethane1.04C−1.060.11X+0.700.15X+0.941,1-Dichloroethene0.98C−0.870.21X−0.230.29X−0.40trans-1,2-Dichloroethene0.97C−0.160.11X+1.460.17X+1.461,2-Dichloropropane a1.00C0.13X0.23Xcis-1,3-Dichloropropene a1.00C0.18X0.32Xtrans-1,3-Dichloropropene a1.00C0.18X0.32XMethylene chloride0.91C−0.930.11X+0.330.21X+1.431,1,2,2-Tetrachloroethene0.95C+0.190.14X+2.410.23X+2.79Tetrachloroethene0.94C+0.060.14X+0.380.18X+2.211,1,1-Trichloroethane0.90C−0.160.15X+0.040.20X+0.371,1,2-Trichloroethane0.86C+0.300.13X−0.140.19X+0.67Trichloroethene0.87C+0.480.13X−0.030.23X+0.30Trichlorofluoromethane0.89C−0.070.15X+0.670.26X+0.91Vinyl chloride0.97C−0.360.13X+0.650.27X+0.40X′=Expected recovery for one or more measurements of a sample containing a concentration of C, in μg/L.sn′=Expected single analyst standard deviation of measurements at an average concentration found of X, in μg/L.S1=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in μg/L.C=True value for the concentration, in μg/L.X=Average recovery found for measurements of samples containing a concentration of C, in μg/L.a Estimates based upon the performance in a single laboratory.10EC02JY92.000EC02JY92.001EC02JY92.002EC02JY92.003Method 602—Purgeable AromaticsPt. 136, App. A, Meth. 6021. Scope and Application

1.1This method covers the determination of various purgeable aromatics. The following parameters may be determined by this method:

1.2This is a purge and trap gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 624 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above.

1.3The method detection limit (MDL, defined in Section 12.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5This method is restricted to use by or under the supervision of analysts experienced in the operation of a purge and trap system and a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1An inert gas is bubbled through a 5-mL water sample contained in a specially-designed purging chamber at ambient temperature. The aromatics are efficiently transferred from the aqueous phase to the vapor phase. The vapor is swept through a sorbent trap where the aromatics are trapped. After purging is completed, the trap is heated and backflushed with the inert gas to desorb the aromatics onto a gas chromatographic column. The gas chromatograph is temperature programmed to separate the aromatics which are then detected with a photoionization detector.2, 3

2.2The method provides an optional gas chromatographic column that may be helpful in resolving the compounds of interest from interferences that may occur.

3. Interferences

3.1Impurities in the purge gas and organic compounds outgassing from the plumbing ahead of the trap account for the majority of contamination problems. The analytical system must be demonstrated to be free from contamination under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3. The use of non-Teflon plastic tubing, non-Teflon thread sealants, or flow controllers with rubber components in the purge and trap system should be avoided.

3.2Samples can be contaminated by diffusion of volatile organics through the septum seal into the sample during shipment and storage. A field reagent blank prepared from reagent water and carried through the sampling and handling protocol can serve as a check on such contamination.

3.3Contamination by carry-over can occur whenever high level and low level samples are sequentially analyzed. To reduce carry-over, the purging device and sample syringe must be rinsed with reagent water between sample analyses. Whenever an unusually concentrated sample is encountered, it should be followed by an analysis of reagent water to check for cross contamination. For samples containing large amounts of water-soluble materials, suspended solids, high boiling compounds or high aromatic levels, it may be necessary to wash the purging device with a detergent solution, rinse it with distilled water, and then dry it in an oven at 105 °C between analyses. The trap and other parts of the system are also subject to contamination; therefore, frequent bakeout and purging of the entire system may be required.

4. Safety

4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 4-6 for the information of the analyst.

4.2The following parameters covered by this method have been tentatively classified as known or suspected, human or mam-malian carcinogens: benzene and 1,4--dichlorobenzene. Primary standards of -these toxic compounds should be prepared -in a hood. A NIOSH/MESA approved -toxic gas respirator should be worn -when the analyst handles high con-centrations of these toxic compounds.

5. Apparatus and Materials

5.1Sampling equipment, for discrete sampling.

5.1.1Vial]25-mL capacity or larger, equipped with a screw cap with a hole in the center (Pierce #13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 °C before use.

5.1.2Septum—Teflon-faced silicone (Pierce -#12722 or equivalent). Deter-gent wash, rinse with tap and distilled -water, and dry at 105°C for 1 h before use.

5.2Purge and trap system—The purge and trap system consists of three separate pieces of equipment: A purging device, trap, and desorber. Several complete systems are now commercially available.

5.2.1The purging device must be designed to accept 5-mL samples with a water column at least 3 cm deep. The gaseous head space between the water column and the trap must have a total volume of less than 15 mL. The purge gas must pass through the water column as finely divided bubbles with a diameter of less than 3 mm at the origin. The purge gas must be introduced no more than 5 mm from the base of the water column. The purging device illustrated in Figure 1 meets these design criteria.

5.2.2The trap must be at least 25 cm long and have an inside diameter of at least 0.105 in.

5.2.2.1The trap is packed with 1 cm of methyl silicone coated packing (Section 6.4.2) and 23 cm of 2,6-diphenylene oxide polymer (Section 6.4.1) as shown in Figure 2. This trap was used to develop the method performance statements in Section 12.

5.2.2.2Alternatively, either of the two traps described in Method 601 may be used, although water vapor will preclude the measurement of low concentrations of benzene.

5.2.3The desorber must be capable of rapidly heating the trap to 180 °C. The polymer section of the trap should not be heated higher than 180 °C and the remaining sections should not exceed 200 °C. The desorber illustrated in Figure 2 meets these design criteria.

5.2.4The purge and trap system may be assembled as a separate unit or be coupled to a gas chromatograph as illustrated in Figures 3, 4, and 5.

5.3Gas chromatograph—An analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.3.1Column 1—6 ft long x 0.082 in. ID stainless steel or glass, packed with 5% SP-1200 and 1.75% Bentone-34 on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statements in Section 12. Guidelines for the use of alternate column packings are provided in Section 10.1.

5.3.3Detector—Photoionization detector (h-Nu Systems, Inc. Model PI-51-02 or equivalent). This type of detector has been proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 12. Guidelines for the use of alternate detectors are provided in Section 10.1.

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.1.1Reagent water can be generated by passing tap water through a carbon filter bed containing about 1 lb of activated carbon (Filtrasorb-300, Calgon Corp., or equivalent).

6.1.2A water purification system (Millipore Super-Q or equivalent) may be used to generate reagent water.

6.1.3Reagent water may also be prepared by boiling water for 15 min. Subsequently, while maintaining the temperature at 90 °C, bubble a contaminant-free inert gas through the water for 1 h. While still hot, transfer the water to a narrow mouth screw-cap bottle and seal with a Teflon-lined septum and cap.

6.6Stock standard solutions—Stock stand-ard solutions may be prepared from pure -standard materials or purchased as certified -solutions. Prepare stock standard solutions in -methanol using assayed liquids. Because -of the toxicity of benzene and 1,4-dichloro-benzene, primary dilutions of these mate-rials should be prepared in a hood. A -NIOSH/MESA approved toxic gas respirator should be used when the analyst handles high concentrations of such materials.

6.6.1Place about 9.8 mL of methanol into a 10-mL ground glass stoppered volumetric flask. Allow the flask to stand, unstoppered, for about 10 min or until all alcohol wetted surfaces have dried. Weigh the flask to the nearest 0.1 mg.

6.6.2Using a 100-μL syringe, immediately add two or more drops of assayed reference material to the flask, then reweigh. Be sure that the drops fall directly into the alcohol without contacting the neck of the flask.

6.6.3Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in μg/μL from the net gain in weight. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.6.4Transfer the stock standard solution into a Teflon-sealed screw-cap bottle. Store at 4 °C and protect from light.

6.6.5All standards must be replaced after one month, or sooner if comparison with check standards indicates a problem.

6.7Secondary dilution standards—Using stock standard solutions, prepare secondary dilution standards in methanol that contain the compounds of interest, either singly or mixed together. The secondary dilution standards should be prepared at concentrations such that the aqueous calibration standards prepared in Section 7.3.1 or 7.4.1 will bracket the working range of the analytical system. Secondary solution standards must be stored with zero headspace and should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.8Quality control check sample concentrate—See Section 8.2.1.

7.Calibration

7.1Assemble a purge and trap system that meets the specifications in Section 5.2. Condition the trap overnight at 180 °C by backflushing with an inert gas flow of at least 20 mL/min. Condition the trap for 10 min once daily prior to use.

7.2Connect the purge and trap system to a gas chromatograph. The gas chromatograph must be operated using temperature and flow rate conditions equivalent to those given in Table 1. Calibrate the purge and trap-gas chromatographic system using either the external standard technique (Section 7.3) or the internal standard technique (Section 7.4).

7.3External standard calibration procedure:

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter by carefully adding 20.0 μL of one or more secondary dilution standards to 100, 500, or 1000 mL of reagent water. A 25-μL syringe with a 0.006 in. ID needle should be used for this operation. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector. These aqueous standards must be prepared fresh daily.

7.3.2Analyze each calibration standard according to Section 10, and tabulate peak height or area responses versus the concentration in the standard. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to concentration (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.4Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples. The compound, α,α,α,-trifluorotoluene, recommended as a surrogate spiking compound in Section 8.7 has been used successfully as an internal standard.

7.4.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest as described in Section 7.3.1.

7.4.2Prepare a spiking solution containing each of the internal standards using the procedures described in Sections 6.6 and 6.7. It is recommended that the secondary dilution standard be prepared at a concentration of 15 μg/mL of each internal standard compound. The addition of 10 μl of this standard to 5.0 mL of sample or calibration standard would be equivalent to 30 μg/L.

7.4.3Analyze each calibration standard according to Section 10, adding 10 μL of internal standard spiking solution directly to the syringe (Section 10.4). Tabulate peak height or area responses against concentration for each compound and internal standard, and calculate response factors (RF) for each compound using Equation 1.

RF=(As)(Cis)(Ais)(Cs)Equation 1where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standardCs=Concentration of the parameter to be measured.If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.5The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of a QC check sample.

7.5.1Prepare the QC check sample as described in Section 8.2.2.

7.5.2Analyze the QC check sample according to Section 10.

7.5.3For each parameter, compare the response (Q) with the corresponding calibration acceptance criteria found in Table 2. If the responses for all parameters of interest fall within the designated ranges, analysis of actual samples can begin. If any individual Q falls outside the range, a new calibration curve, calibration factor, or RF must be prepared for that parameter according to Section 7.3 or 7.4.

8. Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality control program. The mimimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Section 10.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Each day, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system are under control.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 10 μg/mL in methanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.3Analyze four 5-mL aliquots of the well-mixed QC check sample according to Section 10.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for each parameter of interest using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note:

The large number of parameters in Table 2 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.3.

8.2.6.2Beginning with Section 8.2.3, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.3.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 20 μg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.2Analyze one 5-mL sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second 5-mL sample aliquot with 10 μL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than 20 μg/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T) ± 2.44(100 S′/T)%.7

8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 10 μL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 5 mL of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P−2sp to P+2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

8.7The analyst should monitor both the performance of the analytical system and the effectiveness of the method in dealing with each sample matrix by spiking each sample, standard, and reagent water blank with surrogate compounds (e.g. α, α, α,-trifluorotoluene) that encompass the range of the temperature program used in this method. From stock standard solutions prepared as in Section 6.6, add a volume to give 750 μg of each surrogate to 45 mL of reagent water contained in a 50-mL volumetric flask, mix and dilute to volume for a concentration of 15 mg/μL. Add 10 μL of this surrogate spiking solution directly into the 5-mL syringe with every sample and reference standard analyzed. Prepare a fresh surrogate spiking solution on a weekly basis. If the internal standard calibration procedure is being used, the surrogate compounds may be added directly to the internal standard spiking solution (Section 7.4.2).

9. Sample Collection, Preservation, and Handling

9.1The samples must be iced or refrigerated from the time of collection until analysis. If the sample contains free or combined chlorine, add sodium thiosulfate preservative (10 mg/40 mL is sufficient for up to 5 ppm Cl2) to the empty sample bottle just prior to shipping to the sampling site. EPA Method 330.4 or 330.5 may be used for measurement of residual chlorine.8 Field test kits are available for this purpose.

9.2Collect about 500 mL of sample in a clean container. Adjust the pH of the sample to about 2 by adding 1+1 HCl while stirring. Fill the sample bottle in such a manner that no air bubbles pass through the sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. Maintain the hermetic seal on the sample bottle until time of analysis.

9.3All samples must be analyzed within 14 days of collection.3

10. Procedure

10.1Table 1 summarizes the recommended operating conditions for the gas chrom-atograph. Included in this table -are estimated retention times and MDL -that can be achieved under these conditions. An ex-ample of the separations achieved by Column 1 is shown in Figure 6. Other packed columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

10.2Calibrate the system daily as described in Section 7.

10.3Adjust the purge gas (nitrogen or helium) flow rate to 40 mL/min. Attach the trap inlet to the purging device, and set the purge and trap system to purge (Figure 3). Open the syringe valve located on the purging device sample introduction needle.

10.4Allow the sample to come to ambient temperature prior to introducing it to the syringe. Remove the plunger from a 5-mL syringe and attach a closed syringe valve. Open the sample bottle (or standard) and carefully pour the sample into the syringe barrel to just short of overflowing. Replace the syringe plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Since this process of taking an aliquot destroys the validity of the sample for future analysis, the analyst should fill a second syringe at this time to protect against possible loss of data. Add 10.0 μL of the surrogate spiking solution (Section 8.7) and 10.0 μL of the internal standard spiking solution (Section 7.4.2), if applicable, through the valve bore, then close the valve.

10.5Attach the syringe-syringe valve assembly to the syringe valve on the purging device. Open the syringe valves and inject the sample into the purging chamber.

10.6Close both valves and purge the sample for 12.0±0.1 min at ambient temperature.

10.7After the 12-min purge time, disconnect the purging device from the trap. Dry the trap by maintaining a flow of 40 mL/min of dry purge gas through it for 6 min (Figure 4). If the purging device has no provision for bypassing the purger for this step, a dry purger should be inserted into the device to minimize moisture in the gas. Attach the trap to the chromatograph, adjust the purge and trap system to the desorb mode (Figure 5), and begin to temperature program the gas chromatograph. Introduce the trapped materials to the GC column by rapidly heating the trap to 180 °C while backflushing the trap with an inert gas between 20 and 60 mL/min for 4 min. If rapid heating of the trap cannot be achieved, the GC column must be used as a secondary trap by cooling it to 30 °C (subambient temperature, if poor peak geometry and random retention time problems persist) instead of the initial program temperature of 50 °C.

10.8While the trap is being desorbed into the gas chromatograph column, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL flushes of reagent water.

10.9After desorbing the sample for 4 min, recondition the trap by returning the purge and trap system to the purge mode. Wait 15 s, then close the syringe valve on the purging device to begin gas flow through the trap. The trap temperature should be maintained at 180 °C. After approximately 7 min, turn off the trap heater and open the syringe valve to stop the gas flow through the trap. When the trap is cool, the next sample can be analyzed.

10.10Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. -The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

10.11If the response for a peak exceeds the working range of the system, prepare a dilution of the sample with reagent water from the aliquot in the second syringe and reanalyze.

11. Calculations

11.1Determine the concentration of individual compounds in the sample.

11.1.1If the external standard calibration procedure is used, calculate the concentration of the parameter being measured from the peak response using the calibration curve or calibration factor determined in Section 7.3.2.

11.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.4.3 and Equation 2.

EC15NO91.096

Equation 2

where:As = Response for the parameter to be measured.Ais = Response for the internal standard.Cis = Concentration of the internal standard.

11.2Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

12. Method Performance

12.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.9 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

12.2This method has been demonstrated to be applicable for the concentration range from the MDL to 100 X MDL.9 Direct aqueous injection techniques should be used to measure concentration levels above 1000 x MDL.

12.3This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 2.1 to 550 μg/L.9 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3. is two times the value 1.22 derived in this report.)

8.“Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA-600/4-79-020, U.S. Environmental Protection Agency, Office of Research and Development, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268. March 1979.

Table 1—Chromatographic Conditions and Method Detection LimitsParameterRetention time (min)Column 1Column 2Method detection limit (μg/L)Benzene3.332.750.2Toluene5.754.250.2Ethylbenzene8.256.250.2Chlorobenzene9.178.020.21,4-Dichlorobenzene16.816.20.31,3-Dichlorobenzene18.215.00.41,2-Dichlorobenzene25.919.40.4Column 1 conditions: Supelcoport (100/120 mesh) coated with 5% SP-1200/1.75% Bentone-34 packed in a 6 ft x 0.085 in. ID stainless steel column with helium carrier gas at 36 mL/min flow rate. Column temperature held at 50 °C for 2 min then programmed at 6 °C/min to 90 °C for a final hold.Column 2 conditions: Chromosorb W-AW (60/80 mesh) coated with 5% 1,2,3-Tris(2-cyanoethyoxy)propane packed in a 6 ft x 0.085 in. ID stainless steel column with helium carrier gas at 30 mL/min flow rate. Column temperature held at 40 °C for 2 min then programmed at 2 °C/min to 100 °C for a final hold.Table 2—Calibration and QC Acceptance Criteria—Method 602 aParameterRange for Q (μg/L)Limit for s (μg/L)Range for X (μg/L)Range for P, Ps(%)Benzene15.4-24.64.110.0-27.939-150Chlorobenzene16.1-23.93.512.7-25.455-1351,2-Dichlorobenzene13.6-26.45.810.6-27.637-1541,3-Dichlorobenzene14.5-25.55.012.8-25.550-1411,4-Dichlorobenzene13.9-26.15.511.6-25.542-143Ethylbenzene12.6-27.46.710.0-28.232-160Toluene15.5-24.54.011.2-27.746-148Q=Concentration measured in QC check sample, in μg/L (Section 7.5.3).s=Standard deviation of four recovery measurements, in μg/L (Section 8.2.4).X=Average recovery for four recovery measurements, in μg/L (Section 8.2.4).Ps, P=Percent recovery measured (Section 8.3.2, Section 8.4.2).a Criteria were calculated assuming a QC check sample concentration of 20 μg/L.Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.Table 3—Method Accuracy and Precision as Functions of Concentration—Method 602ParameterAccuracy, as recovery, X′ (μg/L)Single analyst precision, s′ (μg/L)Overall precision, S′ (μg/L)Benzene0.92C+0.570.09X+0.590.21X+0.56Chlorobenzene0.95C+0.020.09X+0.230.17X+0.101,2-Dichlorobenzene0.93C+0.520.17X−0.040.22X+0.531,3-Dichlorobenzene0.96C−0.050.15X−0.100.19X+0.091,4-Dichlorobenzene0.93C−0.090.15X+0.280.20X+0.41Ethylbenzene0.94C+0.310.17X+0.460.26X+0.23Toluene0.94C+0.650.09X+0.480.18X+0.71X′=Expected recovery for one or more measurements of a sample containing a concentration of C, in μg/L.S′=Expected single analyst standard deviation of measurements at an average concentration found of X, in X μg/L.S′=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in μg/L.C=True value for the Concentration, in μg/L.X=Average recovery found for measurements of samples containing a concentration of C, in μg/L.EC02JY92.004EC02JY92.005EC02JY92.006EC02JY92.007Method 603—Acrolein and AcrylonitrilePt. 136, App. A, Meth. 6031. Scope and Application

1.1This method covers the determination of acrolein and acrylonitrile. The following parameters may be determined by this method:

1.2This is a purge and trap gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for either or both of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 624 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for the parameters listed above, if used with the purge and trap conditions described in this method.

1.3The method detection limit (MDL, defined in Section 12.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5This method is restricted to use by or under the supervision of analysts experienced in the operation of a purge and trap system and a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1An inert gas is bubbled through a 5-mL water sample contained in a heated purging chamber. Acrolein and acrylonitrile are transferred from the aqueous phase to the vapor phase. The vapor is swept through a sorbent trap where the analytes are trapped. After the purge is completed, the trap is heated and backflushed with the inert gas to desorb the compound onto a gas chromatographic column. The gas chromatograph is temperature programmed to separate the analytes which are then detected with a flame ionization detector.2, 3

2.2The method provides an optional gas chromatographic column that may be helpful in resolving the compounds of interest from the interferences that may occur.

3. Interferences

3.1Impurities in the purge gas and organic compound outgassing from the plumbing of the trap account for the majority of contamination problems. The analytical system must be demonstrated to be free from contamination under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3. The use of non-Teflon plastic tubing, non-Teflon thread sealants, or flow controllers with rubber components in the purge and trap system should be avoided.

3.2Samples can be contaminated by diffusion of volatile organics through the septum seal into the sample during shipment and storage. A field reagent blank prepared from reagent water and carried through the sampling and handling protocol can serve as a check on such contamination.

3.3Contamination by carry-over can occur whenever high level and low level samples are sequentially analyzed. To reduce carry-over, the purging device and sample syringe must be rinsed between samples with reagent water. Whenever an unusually concentrated sample is encountered, it should be followed by an analysis of reagent water to check for cross contamination. For samples containing large amounts of water-soluble materials, suspended solids, high boiling compounds or high analyte levels, it may be necessary to wash the purging device with a detergent solution, rinse it with distilled water, and then dry it in an oven at 105 °C between analyses. The trap and other parts of the system are also subject to contamination, therefore, frequent bakeout and purging of the entire system may be required.

4. Safety

4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this view point, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 4, 6 for the information of the analyst.

5. Apparatus and Materials

5.1Sampling equipment, for discrete sampling.

5.1.1Vial—25-mL capacity or larger, equipped with a screw cap with a hole in the center (Pierce #13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 °C before use.

5.1.2Septum—Teflon-faced silicone (Pierce #12722 or equivalent). Detergent wash, rinse with tap and distilled water and dry at 105 °C for 1 h before use.

5.2Purge and trap system—The purge and trap system consists of three separate pieces of equipment: a purging device, trap, and desorber. Several complete systems are now commercially available.

5.2.1The purging device must be designed to accept 5-mL, samples with a water column at least 3 cm deep. The gaseous head space between the water column and the trap must have a total volume of less than 15 mL. The purge gas must pass through the water column as finely divided bubbles with a diameter of less than 3 mm at the origin. The purge gas must be introduced no more than 5 mm from the base of the water column. The purging device must be capable of being heated to 85 °C within 3.0 min after transfer of the sample to the purging device and being held at 85 ±2 °C during the purge cycle. The entire water column in the purging device must be heated. Design of this modification to the standard purging device is optional, however, use of a water bath is suggested.

5.2.1.1Heating mantle—To be used to heat water bath.

5.2.1.2Temperature controller—Equipped with thermocouple/sensor to accurately control water bath temperature to ±2 °C. The purging device illustrated in Figure 1 meets these design criteria.

5.2.2The trap must be at least 25 cm long and have an inside diameter of at least 0.105 in. The trap must be packed to contain 1.0 cm of methyl silicone coated packing (Section 6.5.2) and 23 cm of 2,6-diphenylene oxide polymer (Section 6.5.1). The minimum specifications for the trap are illustrated in Figure 2.

5.2.3The desorber must be capable of rapidly heating the trap to 180 °C, The desorber illustrated in Figure 2 meets these design criteria.

5.2.4The purge and trap system may be assembled as a separate unit as illustrated in Figure 3 or be coupled to a gas chromatograph.

5.4Gas chromatograph—An analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.4.1Column 1—10 ft long x 2 mm ID glass or stainless steel, packed with Porapak-QS (80/100 mesh) or equivalent. This column was used to develop the method performance statements in Section 12. Guidelines for the use of alternate column packings are provided in Section 10.1.

5.4.3Detector—Flame ionization detector. This type of detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 12. Guidelines for the use of alternate detectors are provided in Section 10.1.

5.5Syringes—5-mL, glass hypodermic with Luerlok tip (two each).

5.6Micro syringes—25-μL, 0.006 in. ID needle.

5.7Syringe valve—2-way, with Luer ends (three each).

5.8Bottle—15-mL, screw-cap, with Teflon cap liner.

5.9Balance—Analytical, capable of accurately weighing 0.0001 g.

6. Reagents

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.1.1Reagent water can be generated by passing tap water through a carbon filter bed containing about 1 lb of activated carbon (Filtrasorb-300, Calgon Corp., or equivalent).

6.1.2A water purification system (Millipore Super-Q or equivalent) may be used to generate reagent water.

6.1.3Regent water may also be prepared by boiling water for 15 min. Subsequently, while maintaining the temperature at 90 °C, bubble a contaminant-free inert gas through the water for 1 h. While still hot, transfer the water to a narrow mouth screw-cap bottle and seal with a Teflon-lined septum and cap.

6.6Stock standard solutions—Stock standard solutions may be prepared from pure standard materials or purchased as certified solutions. Prepare stock standard solutions in reagent water using assayed liquids. Since acrolein and acrylonitrile are lachrymators, primary dilutions of these compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be used when the analyst handles high concentrations of such materials.

6.6.1Place about 9.8 mL of reagent water into a 10-mL ground glass stoppered volumetric flask. For acrolein standards the reagent water must be adjusted to pH 4 to 5. Weight the flask to the nearest 0.1 mg.

6.6.2Using a 100-μL syringe, immediately add two or more drops of assayed reference material to the flask, then reweigh. Be sure that the drops fall directly into the water without contacting the neck of the flask.

6.6.3Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in μg/μL from the net gain in weight. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock staldard. Optionally, stock standard solutions may be prepared using the pure standard material by volumetrically measuring the appropriate amounts and determining the weight of the material using the density of the material. Commercially prepared stock standards may be used at any concentration if they are certified by the manufactaurer or by an independent source.

6.6.4Transfer the stock standard solution into a Teflon-sealed screw-cap bottle. Store at 4 °C and protect from light.

6.6.5Prepare fresh standards daily.

6.7Secondary dilution standards—Using stock standard solutions, prepare secondary dilution standards in reagent water that contain the compounds of interest, either singly or mixed together. The secondary dilution standards should be prepared at concentrations such that the aqueous calibration standards prepared in Section 7.3.1 or 7.4.1 will bracket the working range of the analytical system. Secondary dilution standards should be prepared daily and stored at 4 °C.

6.8Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1Assemble a purge and trap system that meets the specifications in Section 5.2. Condition the trap overnight at 180 °C by backflushing with an inert gas flow of at least 20 mL/min. Condition the trap for 10 min once daily prior to use.

7.2Connect the purge and trap system to a gas chromatograph. The gas chromatograph must be operated using temperature and flow rate conditions equivalent to those given in Table 1. Calibrate the purge and trap-gas chromatographic system using either the external standard technique (Section 7.3) or the internal standard technique (Section 7.4).

7.3External standard calibration procedure:

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter by carefully adding 20.0 μL of one or more secondary dilution standards to 100, 500, or 1000 mL of reagent water. A 25-μL syringe with a 0.006 in. ID needle should be used for this operation. One of the external standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector. These standards must be prepared fresh daily.

7.3.2Analyze each calibration standard according to Section 10, and tabulate peak height or area responses versus the concentration of the standard. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to concentration (calibration factor) is a constant over the working range (< 10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.4Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.4.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest as described in Section 7.3.1.

7.4.2Prepare a spiking solution containing each of the internal standards using the procedures described in Sections 6.6 and 6.7. It is recommended that the secondary dilution standard be prepared at a concentration of 15 μg/mL of each internal standard compound. The addition of 10 μL of this standard to 5.0 mL of sample or calibration standard would be equivalent to 30 μg/L.

7.4.3Analyze each calibration standard according to Section 10, adding 10 μL of internal standard spiking solution directly to the syringe (Section 10.4). Tabulate peak height or area responses against concentration for each compound and internal standard, and calculate response factors (RF) for each compound using Equation 1.

RF=(As)(Cis)(Ais)(Cs)Equation 1where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard.Cs=Concentration of the parameter to be measured.If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.5The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of a QC check sample.

7.5.1Prepare the QC check sample as described in Section 8.2.2.

7.5.2Analyze the QC check sample according to Section 10.

7.5.3For each parameter, compare the response (Q) with the corresponding calibration acceptance criteria found in Table 2. If the responses for all parameters of interest fall within the designated ranges, analysis of actual samples can begin. If any individual Q falls outside the range, a new calibration curve, calibration factor, or RF must be prepared for that parameter according to Section 7.3 or 7.4.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Section 10.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Each day, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system are under control.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 25 μg/mL in reagent water. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.3Analyze four 5-mL aliquots of the well-mixed QC check sample according to Section 10.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If either s exceeds the precision limit or X falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for each compound of interest.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 50 μg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.2Analyze one 5-mL sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second 5-mL sample aliquot with 10 μL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 3. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7

8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 10 μL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 5 mL of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 3. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P−2sp to P+2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1All samples must be iced or refrigerated from the time of collection until analysis. If the sample contains free or combined chlorine, add sodium thiosulfate preservative (10 mg/40 mL is sufficient for up to 5 ppm Cl2) to the empty sample bottle just prior to shipping to the sampling site. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.8 Field test kits are available for this purpose.

9.2If acrolein is to be analyzed, collect about 500 mL of sample in a clean glass container. Adjust the pH of the sample to 4 to 5 using acid or base, measuring with narrow range pH paper. Samples for acrolein analysis receiving no pH adjustment must be analyzed within 3 days of sampling.

9.3Grab samples must be collected in glass containers having a total volume of at least 25 mL. Fill the sample bottle just to overflowing in such a manner that no air bubbles pass through the sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. If preservative has been added, shake vigorously for 1 min. Maintain the hermetic seal on the sample bottle until time of analysis.

9.4All samples must be analyzed within 14 days of collection.3

10. Procedure

10.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are estimated retention times and MDL that can be achieved under these conditions. An example of the separations achieved by Column 1 is shown in Figure 5. Other packed columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

10.2Calibrate the system daily as described in Section 7.

10.3Adjust the purge gas (nitrogen or helium) flow rate to 20 mL-min. Attach the trap inlet to the purging device, and set the purge and trap system to purge (Figure 3). Open the syringe valve located on the purging device sample introduction needle.

10.4Remove the plunger from a 5-mL syringe and attach a closed syringe valve. Open the sample bottle (or standard) and carefully pour the sample into the syringe barrel to just short of overflowing. Replace the syringe plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Since this process of taking an aliquot destroys the validity of the sample for future analysis, the analyst should fill a second syringe at this time to protect against possible loss of data. Add 10.0 μL of the internal standard spiking solution (Section 7.4.2), if applicable, through the valve bore then close the valve.

10.5Attach the syringe-syringe valve assembly to the syringe valve on the purging device. Open the syringe valves and inject the sample into the purging chamber.

10.6Close both valves and purge the sample for 15.0 ± 0.1 min while heating at 85 ± 2 °C.

10.7After the 15-min purge time, attach the trap to the chromatograph, adjust the purge and trap system to the desorb mode (Figure 4), and begin to temperature program the gas chromatograph. Introduce the trapped materials to the GC column by rapidly heating the trap to 180 °C while backflushing the trap with an inert gas between 20 and 60 mL/min for 1.5 min.

10.8While the trap is being desorbed into the gas chromatograph, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL flushes of reagent water.

10.9After desorbing the sample for 1.5 min, recondition the trap by returning the purge and trap system to the purge mode. Wait 15 s then close the syringe valve on the purging device to begin gas flow through the trap. The trap temperature should be maintained at 210 °C. After approximately 7 min, turn off the trap heater and open the syringe valve to stop the gas flow through the trap. When the trap is cool, the next sample can be analyzed.

10.10Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

11. Calculations

11.1Determine the concentration of individual compounds in the sample.

11.1.1If the external standard calibration procedure is used, calculate the concentration of the parameter being measured from the peak response using the calibration curve or calibration factor determined in Section 7.3.2.

11.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.4.3 and Equation 2.

EC15NO91.097Equation 2where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard.

11.2Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

12. Method Performance

12.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.9 The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

12.2This method is recommended for the concentration range from the MDL to 1,000×MDL. Direct aqueous injection techniques should be used to measure concentration levels above 1,000×MDL.

12.3In a single laboratory (Battelle-Columbus), the average recoveries and standard deviations presented in Table 2 were obtained.9 Seven replicate samples were analyzed at each spike level.

1.2This is a flame ionization detector gas chromatographic (FIDGC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for derivatization, cleanup, and electron capture detector gas chromatography (ECDGC) that can be used to confirm measurements made by FIDGC. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3 The method detection limit (MDL, defined in Section 14.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix. The MDL listed in Table 1 for each parameter was achieved with a flame ionization detector (FID). The MDLs that were achieved when the derivatization cleanup and electron capture detector (ECD) were employed are presented in Table 2.

1.4Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1A measured volume of sample, approximately 1-L, is acidified and extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to 2-propanol during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the phenols are then measured with an FID.2

2.2A preliminary sample wash under basic conditions can be employed for samples having high general organic and organic base interferences.

2.3The method also provides for a derivatization and column chromatography cleanup procedure to aid in the elimination of interferences.2,3The derivatives are analyzed by ECDGC.

3. Interferences

3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.4 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are coextracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The derivatization cleanup procedure in Section 12 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Tables 1 and 2.

3.3The basic sample wash (Section 10.2) may cause significantly reduced recovery of phenol and 2,4-dimethylphenol. The analyst must recognize that results obtained under these conditions are minimum concentrations.

4. Safety

4.1The toxicity or carcinogenicity of each reagent used in this mothod has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 5—7 for the information of analyst.

4.2Special care should be taken in handling pentafluorobenzyl bromide, which is a lachrymator, and 18-crown-6-ether, which is highly toxic.

5. Apparatus and Materials

5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle—1-L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.4Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K-570050-1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.4Water bath—Heated, with concentric ring cover, capable of temperature control (±2°C). The bath should be used in a hood.

5.5Balance—Analytical, capable of accurately weighting 0.0001 g.

5.6Gas chromatograph—An analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1Column for underivatized phenols—1.8 m long x 2 mm ID glass, packed with 1% SP-1240DA on Supelcoport (80/100 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 11.1.

5.6.2Column for derivatized phenols—1.8 m long x 2 mm ID glass, packed with 5% OV-17 on Chromosorb W-AW-DMCS (80/100 mesh) or equivalent. This column has proven effective in the analysis of wastewaters for derivatization products of the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 11.1.

5.6.3Detectors—Flame ionization and electron capture detectors. The FID is used when determining the parent phenols. The ECD is used when determining the derivatized phenols. Guidelines for the use of alternatve detectors are provided in Section 11.1.

6. Reagents

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.11Derivatization reagent—Add 1 mL of pentafluorobenzyl bromide and 1 g of 18-crown-6-ether to a 50-mL volumetric flask and dilute to volume with 2-propanol. Prepare fresh weekly. This operation should be carried out in a hood. Store at 4 °C and protect from light.

6.14.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in 2-propanol and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.14.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.14.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.15Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1To calibrate the FIDGC for the anaylsis of underivatized phenols, establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure for FIDGC:

7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with 2-propanol. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 μl, analyze each calibration standard according to Section 11 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3Internal standard calibration procedure for FIDGC—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with 2-propanol. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 11 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

RF=(As)(Cis)(Ais)(Cs)Equation 1where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard (μg/L).Cs=Concentration of the parameter to be measured (μg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound.

7.5To calibrate the ECDGC for the analysis of phenol derivatives, establish gas chromatographic operating conditions equivalent to those given in Table 2.

7.5.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with 2-propanol. One of the external standards should be at a concentration near, but above, the MDL (Table 2) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.5.2Each time samples are to be derivatized, simultaneously treat a 1-mL aliquot of each calibration standard as described in Section 12.

7.5.3After derivatization, analyze 2 to 5 μL of each column eluate collected according to the method beginning in Section 12.8 and tabulate peak height or area responses against the calculated equivalent mass of underivatized phenol injected. The results can be used to prepare a calibration curve for each compound.

7.6Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.6 and 11.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 100 μg/mL in 2-propanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at a concentration of 100 μg/L by adding 1.00 mL of QC check sample concentrate to each of four 1-L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note:

The large number of parameters in Talbe 3 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.2.6.2Beginning with Section 8.2.2, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 100 μg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any, or, if none, (2) the larger of either 5 times higher than the expected background concentration or 100 μg/L.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 3. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.8 If spiking was performed at a concentration lower than 100 μg/L, the analyst must use either the QC acceptance criteria in Table 3, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 4, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 4, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.8

8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 3. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P−2sp to P+2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6.It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1Grab samples must be collected in glass containers. Conventional sampling practices 9 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 °C from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.10 Field test kits are available for this purpose.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1Mark the water meniscus on the side of sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel.

10.2For samples high in organic content, the analyst may solvent wash the sample at basic pH as prescribed in Sections 10.2.1 and 10.2.2 to remove potential method interferences. Prolonged or exhaustive contact with solvent during the wash may result in low recovery of some of the phenols, notably phenol and 2,4-dimethylphenol. For relatively clean samples, the wash should be omitted and the extraction, beginning with Section 10.3, should be followed.

10.2.1Adjust the pH of the sample to 12.0 or greater with sodium hydroxide solution.

10.2.2Add 60 mL of methylene chloride to the sample by shaking the funnel for 1 min with periodic venting to release excess pressure. Discard the solvent layer. The wash can be repeated up to two additional times if significant color is being removed.

10.3Adjust the sample to a pH of 1 to 2 with sulfuric acid.

10.4Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.5Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.6Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.7Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.8Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.9Increase the temperature of the hot water bath to 95 to 100 °C. Remove the Synder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of 2-propanol. A 5-mL syringe is recommended for this operation. Attach a two-ball micro-Snyder column to the concentrator tube and prewet the column by adding about 0.5 mL of 2-propanol to the top. Place the micro-K-D apparatus on the water bath so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches 2.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Add an additional 2 mL of 2-propanol through the top of the micro-Snyder column and resume concentrating as before. When the apparent volume of liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.10Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of 2-propanol. Adjust the extract volume to 1.0 mL. Stopper the concentrator tube and store refrigerated at 4 °C if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with FIDGC analysis (Section 11). If the sample requires further cleanup, proceed to Section 12.

10.11Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Flame Ionization Detector Gas Chromatography

11.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. An example of the separations achieved by this column is shown in Figure 1. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

11.2Calibrate the system daily as described in Section 7.

11.3If the internal standard calibration procedure is used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

11.4Inject 2 to 5 μL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.11 Smaller (1.0 μL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 μL, and the resulting peak size in area or peak height units.

11.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound may be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

11.6If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

11.7If the measurement of the peak response is prevented by the presence of interferences, an alternative gas chromatographic procedure is required. Section 12 describes a derivatization and column chromatographic procedure which has been tested and found to be a practical means of analyzing phenols in complex extracts.

12. Derivatization and Electron Capture Detector Gas Chromatography

12.1Pipet a 1.0-mL aliquot of the 2-propanol solution of standard or sample extract into a glass reaction vial. Add 1.0 mL of derivatizing reagent (Section 6.11). This amount of reagent is sufficient to derivatize a solution whose total phenolic content does not exceed 0.3 mg/mL.

12.2Add about 3 mg of potassium carbonate to the solution and shake gently.

12.3Cap the mixture and heat it for 4 h at 80 °C in a hot water bath.

12.4Remove the solution from the hot water bath and allow it to cool.

12.5Add 10 mL of hexane to the reaction flask and shake vigorously for 1 min. Add 3.0 mL of distilled, deionized water to the reaction flask and shake for 2 min. Decant a portion of the organic layer into a concentrator tube and cap with a glass stopper.

12.6Place 4.0 g of silica gel into a chromatographic column. Tap the column to settle the silica gel and add about 2 g of anhydrous sodium sulfate to the top.

12.7Preelute the column with 6 mL of hexane. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, pipet onto the column 2.0 mL of the hexane solution (Section 12.5) that contains the derivatized sample or standard. Elute the column with 10.0 mL of hexane and discard the eluate. Elute the column, in order, with: 10.0 mL of 15% toluene in hexane (Fraction 1); 10.0 mL of 40% toluene in hexane (Fraction 2); 10.0 mL of 75% toluene in hexane (Fraction 3); and 10.0 mL of 15% 2-propanol in toluene (Fraction 4). All elution mixtures are prepared on a volume: volume basis. Elution patterns for the phenolic derivatives are shown in Table 2. Fractions may be combined as desired, depending upon the specific phenols of interest or level of interferences.

12.8Analyze the fractions by ECDGC. Table 2 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. An example of the separations achieved by this column is shown in Figure 2.

12.9Calibrate the system daily with a minimum of three aliquots of calibration standards, containing each of the phenols of interest that are derivatized according to Section 7.5.

12.10Inject 2 to 5 μL of the column fractions into the gas chromatograph using the solvent-flush technique. Smaller (1.0 μL) volumes can be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 μL, and the resulting peak size in area or peak height units. If the peak response exceeds the linear range of the system, dilute the extract and reanalyze.

13. Calculations

13.1Determine the concentration of individual compounds in the sample analyzed by FIDGC (without derivatization) as indicated below.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

EC15NO91.098Equation 2where:A=Amount of material injected (ng).Vi=Volume of extract injected (μL).Vt=Volume of total extract (μL).Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

EC15NO91.099Equation 3where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Is=Amount of internal standard added to each extract (μg).Vo=Volume of water extracted (L).

13.2Determine the concentration of individual compounds in the sample analyzed by derivatization and ECDGC according to Equation 4.

EC15NO91.100Equation 4where:A=Mass of underivatized phenol represented by area of peak in sample chromatogram, determined from calibration curve in Section 7.5.3 (ng).Vi=Volume of eluate injected (μL).Vt=Total volume of column eluate or combined fractions from which Vi was taken (μL).Vs=Volume of water extracted in Section 10.10 (mL).B=Total volume of hexane added in Section 12.5 (mL).C=Volume of hexane sample solution added to cleanup column in Section 12.7 (mL).D=Total volume of 2-propanol extract prior to derivatization (mL).E=Volume of 2-propanol extract carried through derivatization in Section 12.1 (mL).

13.3Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Tables 1 and 2 were obtained using reagent water.12Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked as six concentrations over the range 12 to 450 μg/L.13 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships for a flame ionization detector are presented in Table 4.

4. ASTM Annual Book of Standards, Part 31, D3694-78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

5. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77-206, August 1977.

8. Provost, L. P., and Elder, R. S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

9. ASTM Annual Book of Standards, Part 31, D3370-76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

10. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methmds for Chemical Analysis of Water and Wastes, EPA-600/4-79-020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

1.2This is a high performance liquid chromatography (HPLC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for the compounds above, identifications should be supported by at least one additional qualitative technique. This method describes electrochemical conditions at a second potential which can be used to confirm measurements made with this method. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for the parameters listed above, using the extract produced by this method.

1.3The method detection limit (MDL, defined in Section 14.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of the interferences in the sample matrix.

1.4Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5This method is restricted to use by or under the supervision of analysts experienced in the use of HPLC instrumentation and in the interpretation of liquid chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1A measured volume of sample, approximately 1-L, is extracted with chloroform using liquid-liquid extractions in a separatory funnel. The chloroform extract is extracted with acid. The acid extract is then neutralized and extracted with chloroform. The final chloroform extract is exchanged to methanol while being concentrated using a rotary evaporator. The extract is mixed with buffer and separated by HPLC. The benzidine compounds are measured with an electrochemical detector.2

2.2The acid back-extraction acts as a general purpose cleanup to aid in the elimination of interferences.

3. Interferences

3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.3 Clean all glassware as soon as -possible after use by rinsing with the last -solvent used in it. Solvent rinsing should -be followed by detergent washing with hot -water, and rinses with tap water and -distilled water. The glassware should then be -drained dry, and heated in a muffle furnace at -400 °C for 15 to 30 min. Some thermally -stable materials may not be eliminated by -this treatment. Solvent rinses with acetone -and pesticide quality hexane may be substi-tuted for the muffle furnace heating. Vol-umetric ware should not be heated in a muffle -furnace. After drying and cooling, glass-ware should be sealed and stored in a clean en-vironment to prevent any accumulation of -dust or other contaminants. Store inverted or -capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality -being sampled. The cleanup procedures -that are inherent in the extraction step are -used to overcome many of these interfer-ences, but unique samples may require addi-tional cleanup approaches to achieve -the MDL listed in Table 1.

3.3Some dye plant effluents contain -large amounts of components with re-tention times closed to benzidine. In -these cases, it has been found useful to reduce the electrode potential in order to eliminate interferences and still detect benzidine. (See Section 12.7.)

4. Safety

4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health harzard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 4-6 for the information of the analyst.

4.2The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: benzidine and 3,3′-dichlorobenzidine. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

4.3Exposure to chloroform should be minimized by performing all extractions and extract concentrations in a hood or other well-ventiliated area.

5. Apparatus and Materials

5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle—1-L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4°C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.4.3Electrochemical detector—Bioanalyti-cal Systems LC-2A with glassy carbon -electrode, or equivalent. This detector has -proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1.

5.4.5Column—Lichrosorb RP-2, 5 micron particle diameter, in a 25 cm × 4.6 mm ID stainless steel column. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

6. Reagents

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.8Acetate buffer (0.1 M, pH 4.7)—Dissolve 5.8 mL of glacial acetic acid (ACS) and 13.6 g of sodium acetate trihydrate (ACS) in reagent water which has been purified by filtration through a RO-4 Millipore System or equivalent and dilute to 1 L.

6.11.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in methanol and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.11.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.11.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.12Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1Establish chromatographic operating conditions equivalent to those given in Table 1. The HPLC system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure:

7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with mobile phase. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using syringe injections of 5 to 25 μL or a constant volume injection loop, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with mobile phase. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using syringe injections of 5 to 25 μL or a constant volume injection loop, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

RF=(As)(Cis)(Ais)(Cs)Equation 1where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard (μg/L).Cs=Concentration of the parameter to be measured (μg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound. If serious loss of response occurs, polish the electrode and recalibrate.

7.5Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.9, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing benzidine and/or 3,3′-dichlorobenzidine at a concentration of 50 μg/mL each in methanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at a concentration of 50 μg/L by adding 1.00 mL of QC check sample concentrate to each of four 1-L-L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 50 μg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or 50 μg/L.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than 50 μg/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.7

8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note: The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Sections 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P−2sp to P+2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as HPLC with a dissimilar column, gas chromatography, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1Grab samples must be collected in glass containers. Conventional sampling practices8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4°C and stored in the dark from the time of collection until extraction. Both benzidine and 3,3′-dichlorobenzidine are easily oxidized. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.9 Field test kits are available for this purpose. After mixing, adjust the pH of the sample to a range of 2 to 7 with sulfuric acid.

9.3If 1,2-diphenylhydrazine is likely to be present, adjust the pH of the sample to 4.0± 0.2 to prevent rearrangement to benzidine.

9.4All samples must be extracted within 7 days of collection. Extracts may be held up to 7 days before analysis, if stored under an inert (oxidant free) atmosphere.2 The extract should be protected from light.

10. Sample Extraction

10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of the sample with wide-range pH paper and adjust to within the range of 6.5 to 7.5 with sodium hydroxide solution or sulfuric acid.

10.2Add 100 mL of chloroform to the sample bottle, seal, and shake 30 s to rinse the inner surface. (Caution: Handle chloroform in a well ventilated area.) Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the chloroform extract in a 250-mL separatory funnel.

10.3Add a 50-mL volume of chloroform to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the separatory funnel. Perform a third extraction in the same manner.

10.4Separate and discard any aqueous layer remaining in the 250-mL separatory funnel after combining the organic extracts. Add 25 mL of 1 M sulfuric acid and extract the sample by shaking the funnel for 2 min. Transfer the aqueous layer to a 250-mL beaker. Extract with two additional 25-mL portions of 1 M sulfuric acid and combine the acid extracts in the beaker.

10.5Place a stirbar in the 250-mL beaker and stir the acid extract while carefully adding 5 mL of 0.4 M sodium tribasic phosphate. While monitoring with a pH meter, neutralize the extract to a pH between 6 and 7 by dropwise addition of 5 N sodium hydroxide solution while stirring the solution vigorously. Approximately 25 to 30 mL of 5 N sodium hydroxide solution will be required and it should be added over at least a 2-min period. Do not allow the sample pH to exceed 8.

10.6Transfer the neutralized extract into a 250-mL separatory funnel. Add 30 mL of chloroform and shake the funnel for 2 min. Allow the phases to separate, and transfer the organic layer to a second 250-mL separatory funnel.

10.7Extract the aqueous layer with two additional 20-mL aliquots of chloroform as before. Combine the extracts in the 250-mL separatory funnel.

10.8Add 20 mL of reagent water to the combined organic layers and shake for 30 s.

10.9Transfer the organic extract into a 100-mL round bottom flask. Add 20 mL of methanol and concentrate to 5 mL with a rotary evaporator at reduced pressure and 35 °C. An aspirator is recommended for use as the source of vacuum. Chill the receiver with ice. This operation requires approximately 10 min. Other concentration techniques may be used if the requirements of Section 8.2 are met.

10.10Using a 9-in. Pasteur pipette, transfer the extract to a 15-mL, conical, screw-cap centrifuge tube. Rinse the flask, including the entire side wall, with 2-mL portions of methanol and combine with the original extract.

10.11Carefully concentrate the extract to 0.5 mL using a gentle stream of nitrogen while heating in a 30 °C water bath. Dilute to 2 mL with methanol, reconcentrate to 1 mL, and dilute to 5 mL with acetate buffer. Mix the extract thoroughly. Cap the centrifuge tube and store refrigerated and protected from light if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with HPLC analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.12Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1,000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

12. High Performance Liquid Chromatography

12.1Table 1 summarizes the recommended operating conditions for the HPLC. Included in this table are retention times, capacity factors, and MDL that can be achieved under these conditions. An example of the separations achieved by this HPLC column is shown in Figure 1. Other HPLC columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met. When the HPLC is idle, it is advisable to maintain a 0.1 mL/min flow through the column to prolong column life.

12.2Calibrate the system daily as described in Section 7.

12.3If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the instrument.

12.4Inject 5 to 25 μL of the sample extract or standard into the HPLC. If constant volume injection loops are not used, record the volume injected to the nearest 0.05 μL, and the resulting peak size in area or peak height units.

12.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6If the response for a peak exceeds the working range of the system, dilute the extract with mobile phase and reanalyze.

12.7If the measurement of the peak response for benzidine is prevented by the presence of interferences, reduce the electrode potential to +0.6 V and reanalyze. If the benzidine peak is still obscured by interferences, further cleanup is required.

13. Calculations

13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

EC15NO91.101Equation 2where:A=Amount of material injected (ng).Vi=Volume of extract injected (μL).Vt=Volume of total extract (μL).Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

EC15NO91.102Equation 3where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Is=Amount of internal standard added to each extract (μg).Vo=Volume of water extracted (L).

13.2Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.10 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 7×MDL to 3000×MDL.10

14.3This method was tested by 17 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 70 μg/L.11 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

3. ASTM Annual Book of Standards, Part 31, D3694-78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

4. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77-206, August 1977.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

8. ASTM Annual Book of Standards, Part 31, D3370-76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

9. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA-600/4-79-020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

Table 1—Chromatographic Conditions and Method Detection LimitsParameterRetention time (min)Column capacity factor (k′)Method detection limit (μg/L)Benzidine6.11.440.083,3′-Dichlorobenzidine12.13.840.13HPLC Column conditions: Lichrosorb RP-2, 5 micron particle size, in a 25 cm×4.6 mm ID stainless steel column. Mobile Phase: 0.8 mL/min of 50% acetonitrile/50% 0.1M pH 4.7 acetate buffer. The MDL were determined using an electrochemical detector operated at +0.8 V.Table 2—QC Acceptance Criteria—Method 605ParameterTest conc. (μg/L)Limit for s (μg/L)Range for X (μg/L)Range for P, Ps (percent)Benzidine5018.79.1-61.0D-1403.3′-Dichlorobenzidine5023.618.7-50.05-128s=Standard deviation of four recovery measurements, in μg/L (Section 8.2.4).X=Average recovery for four recovery measurements, in μg/L (Section 8.2.4).P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).D=Detected; result must be greater than zero.Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.Table 3—Method Accuracy and Precision as Functions of Concentration—Method 605ParameterAccuracy, as recovery, X′(μg/L)Single analyst precision, sr′ (μg/L)Overall precision, S′ (μg/L)Benzidine0.70C+0.060.28X+0.190.40X+0.183,3′-Dichlorobenzidine0.66C+0.230.39X−0.050.38X+0.02X′=Expected recovery for one or more measurements of a sample containing a concentration of C, in μg/L.sr′=Expected single analyst standard deviation of measurements at an average concentration found of X, in μg/L.S′=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in μg/L.C=True value for the concentration, in μg/L.X=Average recovery found for measurements of samples containing a concentration of C, in μg/L.EC02JY92.014Method 606—Phthalate EsterPt. 136, App. A, Meth. 6061. Scope and Application

1.1This method covers the determination of certain phthalate esters. The following parameters can be determined by this method:

1.2This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4The sample extraction and concentration steps in this method are essentially the same as in Methods 608, 609, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1A measured volume of sample, approximately 1-L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the phthalate esters are then measured with an electron capture detector.2

2.2Analysis for phthalates is especially complicated by their ubiquitous occurrence in the environment. The method provides Florisil and alumina column cleanup procedures to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.3 Clean all glassware -as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Phthalate esters are contaminants in many products commonly found in the laboratory. It is particularly important to avoid the use of plastics because phthalates are commonly used as plasticizers and are easily extracted from plastic materials. Serious phthalate contamination can result at any time, if consistent quality control is not practiced. Great care must be experienced to prevent such contamination. Exhaustive cleanup of reagents and glassware may be required to eliminate background phthalate contamination.4,5

3.3Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedures in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

4. Safety

4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 6-8 for the information of the analyst.

5. Apparatus and Materials

5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle—1-L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only).

5.2.4Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K-570050-1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.4Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5Balance—Analytical, capable of accurately weighing 0.0001 g.

5.6Gas chromatograph—An analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1Column 1—1.8 m long × 4 mm ID glass, packed with 1.5% SP-2250/1.95% SP-2401 Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statemelts in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.3Detector—Electron capture detector. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1.

6. Reagents

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.3.1Ethyl ether must be shown to be free of peroxides before it is used as indicated by EM Laboratories Quant test strips. (Available from Scientific Products Co., Cat. No. P1126-8, and other suppliers.)

6.3.2Procedures recommended for removal of peroxides are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.

6.4Sodium sulfate—(ACS) Granular, anhydrous. Several levels of purification may be required in order to reduce background phthalate levels to an acceptable level: 1) Heat 4 h at 400 °C in a shallow tray, 2) Heat 16 h at 450 to 500 °C in a shallow tray, 3) Soxhlet extract with methylene chloride for 48 h.

6.5Florisil—PR grade (60/100 mesh). Purchase activated at 1250 °F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. To prepare for use, place 100 g of Florisil into a 500-mL beaker and heat for approximately 16 h at 40 °C. After heating transfer to a 500-mL reagent bottle. Tightly seal and cool to room temperature. When cool add 3 mL of reagent water. Mix thoroughly by shaking or rolling for 10 min and let it stand for at least 2 h. Keep the bottle sealed tightly.

6.6Alumina—Neutral activity Super I, W200 series (ICN Life Sciences Group, No. 404583). To prepare for use, place 100 g of alumina into a 500-mL beaker and heat for approximately 16 h at 400 °C. After heating transfer to a 500-mL reagent bottle. Tightly seal and cool to room temperature. When cool add 3 mL of reagent water. Mix thoroughly by shaking or rolling for 10 min and let it stand for at least 2 h. Keep the bottle sealed tightly.

6.7.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in isooctane and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.8Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1Establish gas chromatograph operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure:

7.2.1Prepared calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with isooctane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flash. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with isooctane. One of the standards should be at a concentraton near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

RF=(As)(Cis)(Ais)(Cs)Equation 1where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard (μg/L).Cs=Concentration of the parameter to be measured (μg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound.

7.5Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality contrml (QC) check sample concentrate is required containing each parameter of interest at the following concentrations in acetone: butyl benzyl phthalate, 10 μg/mL; bis(2-ethylhexyl) phthalate, 50 μg/mL; di-n-octyl phthalate, 50 μg/mL; any other phthlate, 25 μg/mL. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agancy, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at the test concentrations shown in Table 2 by adding 1.00 mL of QC check sample concentrate to each of four 1-L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.9 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.9

8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P−2sp to P+2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1Grab samples must be collected in glass containers. Conventional sampling practices10 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 °C from the time of collection until extraction.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel.

10.2Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phrase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentrator devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.7Increase the temperature of the hot water bath to about 80 °C. Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Concentrate the extract as in Section 10.6, except use hexane to prewet the column. The elapsed time of concentration should be 5 to 10 min.

10.8Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Adjust the extract volume to 10 mL. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.9Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use either procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2If the entire extract is to be cleaned up by one of the following procedures, it must be concentrated to 2.0 mL. To the concentrator tube in Section 10.8, add a clean boiling chip and attach a two-ball micro-Snyder column. Prewet the column by adding about 0.5 mL of hexane to the top. Place the micro-K-D apparatus on a hot water bath (80 °C) so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with 0.2 mL of hexane. Adjust the final volume to 2.0 mL and proceed with one of the following cleanup procedures.

11.3Florisil column cleanup for phthalate esters:

11.3.1Place 10 g of Florisil into a chromatographic column. Tap the column to settle the Florisil and add 1 cm of anhydrous sodium sulfate to the top.

11.3.2Preelute the column with 40 mL of hexane. The rate for all elutions should be about 2 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 2-mL sample extract onto the column using an additional 2 mL of hexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 40 mL of hexane and continue the elution of the column. Discard this hexane eluate.

11.3.3Next, elute the column with 100 mL of 20% ethyl ether in hexane (V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction as in Section 10.6. No solvent exchange is necessary. Adjust the volume of the cleaned up extract to 10 mL in the concentrator tube and analyze by gas chromatography (Section 12).

11.4Alumina column cleanup for phthalate esters:

11.4.1Place 10 g of alumina into a chromatographic column. Tap the column to settle the alumina and add 1 cm of anhydrous sodium sulfate to the top.

11.4.2Preelute the column with 40 mL of hexane. The rate for all elutions should be about 2 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 2-mL sample extract onto the column using an additional 2 mL of hexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 35 mL of hexane and continue the elution of the column. Discard this hexane eluate.

11.4.3Next, elute the column with 140 mL of 20% ethyl ether in hexane (V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator type. Concentrate the collected fraction as in Section 10.6. No solvent exchange is necessary. Adjust the volume of the cleaned up extract to 10 mL in the concentrator tube and analyze by gas chromatography (Section 12).

12. Gas Chromatography

12.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Column 1 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2Calibrate the system daily as described in Section 7.

12.3If the internal standard calibration procedure is being used, the internal staldard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

12.4Inject 2 to 5 μL of the sample extract or standard into the gas-chromatograph using the solvent-flush technique.11 Smaller (1.0 μL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 μL, and the resulting peak size in area or peak height units.

12.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations

13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

EC15NO91.103Equation 2where:A=Amount of material injected (ng).Vi=Volume of extract injected (μL).Vt=Volume of total extract (μL).Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

EC15NO91.104Equation 3where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Is=Amount of internal standard added to each extract (μg).Vo=Volume of water extracted (L).

13.2Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.12 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 5 × MDL to 1000 × MDL with the following exceptions: dimethyl and diethyl phthalate recoveries at 1000 × MDL were low (70%); bis-2-ethylhexyl and di-n-octyl phthalate recoveries at 5 × MDL were low (60%).12

14.3This method was tested by 16 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 0.7 to 106 μg/L.13 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

3. ASTM Annual Book of Standards, Part 31, D3694-78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

9. Provost L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

10. ASTM Annual Book of Standards, Part 31, D3370-76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

1.2This is a gas chromatographic (GC) method applicable to the determination of the parameters listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compmunds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditimns for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for N-nitrosodi-n-propylamine. In order to confirm the presence of N-nitrosodiphenylamine, the cleanup procedure specified in Section 11.3 or 11.4 must be used. In order to confirm the presence of N-nitrosodimethylamine by GC/MS, Column 1 of this method must be substituted for the column recommended in Method 625. Confirmation of these parameters using GC-high resolution mass spectrometry or a Thermal Energy Analyzer is also recommended. 1,2

1.3The method detection limit (MDL, defined in Section 14.1)3 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.5This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1A measured volume of sample, approximately 1-L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is washed with dilute hydrochloric acid to remove free amines, dried, and concentrated to a volume of 10 mL or less. After the extract has been exchanged to methanol, it is separated by gas chromatography and the parameters are then measured with a nitrogen-phosphorus detector.4

2.2The method provides Florisil and alumina column cleanup procedures to separate diphenylamine from the nitrosamines and to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.5 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedures in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

3.3N-Nitrosodiphenylamine is reported6-9 to undergo transnitrosation reactions. Care must be exercised in the heating or concentrating of solutions containing this compound in the presence of reactive amines.

3.4The sensitive and selective Thermal Energy Analyzer and the reductive Hall detector may be used in place of the nitrogen-phosphorus detector when interferences are encountered. The Thermal Energy Analyzer offers the highest selectivity of the non-MS detectors.

4. Safety

4.1The toxicity or carcinogenicity of each -reagent used in this method has not been -precisely defined; however, each chemical -compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 10-12 for the information of the analyst.

4.2These nitrosamines are known carcinogens 13-17, therefore, utmost care must be exercised in the handling of these materials. Nitrosamine reference standards and standard solutions should be handled and prepared in a ventilated glove box within a properly ventilated room.

5. Apparatus and Materials

5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle—1-L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flowmeter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.3Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K-570050-1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.4Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5Balance—Analytical, capable of accurately weighing 0.0001 g.

5.6Gas chromatograph—An analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1Column 1—1.8 m long × 4 mm ID glass, packed with 10% Carbowax 20 M/2% KOH on Chromosorb W-AW (80/100 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.2.

5.6.3Detector—Nitrogen-phosphorus, reductive Hall, or Thermal Energy Analyzer detector.1,2 These detectors have proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1). A nitrogen-phosphorus detector was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.2.

6. Reagents

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.8.1Ethyl ether must be shown to be free of peroxides before it is used as indicated by EM Laboratories Quant test strips. (Available from Scientific Products Co., Cat No. P1126-8, and other suppliers.)

6.8.2Procedures recommended for removal of peroxides are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.

6.9Florisil—PR grade (60/100 mesh). Purchase activated at 1250 °F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 130 °C in a foil-covered glass container and allow to cool.

6.10Alumina—Basic activity Super I, W200 series (ICN Life Sciences Group, No. 404571, or equivalent). To prepare for use, place 100 g of alumina into a 500-mL reagent bottle and add 2 mL of reagent water. Mix the alumina preparation thoroughly by shaking or rolling for 10 min and let it stand for at least 2 h. The preparation should be homogeneous before use. Keep the bottle sealed tightly to ensure proper activity.

6.11.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in methanol and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.11.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.11.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.12Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure:

7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with methanol. One of the external standards should be at a concentraton near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with methanol. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

RF=(As)(Cis)(Ais)(Cs)Equation 1where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard (μg/L).Cs=Concentration of the parameter to be measured (μg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound.

7.5Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.2) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 20 μg/mL in methanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at a concentration of 20 μg/L by adding 1.00 mL of QC check sample concentrate to each of four 1-L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 20 μg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or 20 μg/L.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were caluclated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.18 If spiking was performed at a concentration lower than 20 μg/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria caluclated for the specific spike concentration. To calculate optional acceptance crtieria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T) ± 2.44(100 S′/T)%.18

8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P−2sp to P+2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such -as gas chromatography with a dissimilar -column, specific element detector, or mass -spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1Grab samples must be collected in glass -containers. Conven-tional sampling practices 19 should be -followed, except that the bottle must not be pre-rinsed with sample before collection. Composite sam-ples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 °C from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.20 Field test kits are available for this purpose. If N-nitrosodiphenylamine is to be determined, adjust the sample pH to 7 to 10 with sodium hydroxide solution or sulfuric acid.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.4

9.4Nitrosamines are known to be light sen-sitive.7 Samples should be stored in amber or foil-wrapped bottles in order to minimize photolytic decomposition.

10. Sample Extraction

10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of the sample with wide-range pH paper and adjust to within the range of 5 to 9 with sodium hydroxide solution or sulfuric acid.

10.2Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) con-centrator by attaching a 10-mL concentra-tor tube to a 500-mL evaporative flask. Other -concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Add 10 mL of hydrochloric acid to the combined extracts and shake for 2 min. Allow the layers to separate. Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65°C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.7Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If N-nitro-sodi-phe-nylamine is to be measured by gas chromatography, the analyst must first use a cleanup column to eliminate diphenylamine interference (Section 11). If N-nitrosodiphenylamine is of no interest, the analyst may proceed directly with gas chromatographic analysis (Section 12).

10.8Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-

mL graduated cylinder. Record the sample volume to the nearest 5 mL.11. Cleanup and Separation

11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use either procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure. Diphenylamine, if present in the original sample extract, must be separated from the nitrosamines if N-nitrosodiphenylamine is to be determined by this method.

11.2If the entire extract is to be cleaned up by one of the following procedures, it must be concentrated to 2.0 mL. To the concentrator tube in Section 10.7, add a clean boiling chip and attach a two-ball micro-Snyder column. Prewet the column by adding about 0.5 mL of methylene chloride to the top. Place the micr-K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with 0.2 mL of methylene chloride. Adjust the final volume to 2.0 mL and proceed with one of the following cleanup procedures.

11.3Florisil column cleanup for nitro-samines:

11.3.1Place 22 g of activated Florisil into a 22-mm ID chromatographic column. Tap the column to settle the Florisil and add about 5 mm of anhydrous sodium sulfate to the top.

11.3.2Preelute the column with 40 mL of ethyl ether/pentane (15+85)(V/V). Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 2-mL sample extract onto the column using an additional 2 mL of pentane to complete the transfer.

11.3.3Elute the column with 90 mL of ethyl ether/pentane (15+85)(V/V) and discard the eluate. This fraction will contain the diphenylamine, if it is present in the extract.

11.3.4Next, elute the column with 100 mL of acetone/ethyl ether (5+95)(V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. This fraction will contain all of the nitrosamines listed in the scope of the method.

11.3.5Add 15 mL of methanol to the collected fraction and concentrate as in Section 10.6, except use pentane to prewet the column and set the water bath at 70 to 75 °C. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of pentane. Analyze by gas chromatography (Section 12).

11.4Alumina column cleanup for nitro-samines:

11.4.1Place 12 g of the alumina preparation (Section 6.10) into a 10-mm ID chromatographic column. Tap the column to settle the alumina and add 1 to 2 cm of anhydrous sodium sulfate to the top.

11.4.2Preelute the column with 10 mL of ethyl ether/pentane (3+7)(V/V). Discard the eluate (about 2 mL) and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 2 mL sample extract onto the column using an additional 2 mL of pentane to complete the transfer.

11.4.3Just prior to exposure of the sodium sulfate layer to the air, add 70 mL of ethyl ether/pentane (3+7)(V/V). Discard the first 10 mL of eluate. Collect the remainder of the eluate in a 500-mL K-D flask equipped with a 10 mL concentrator tube. This fraction contains N-nitrosodiphenylamine and probably a small amount of N-nitrosodi-n-propylamine.

11.4.4Next, elute the column with 60 mL of ethyl ether/pentane (1+1)(V/V), collecting the eluate in a second K-D flask equipped with a 10-mL concentrator tube. Add 15 mL of methanol to the K-D flask. This fraction will contain N-nitrosodimethylamine, most of the N-nitrosodi-n-propylamine and any diphenylamine that is present.

11.4.5Concentrate both fractions as in Section 10.6, except use pentane to prewet the column. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of pentane. Analyze the fractions by gas chromatography (Section 12).

12. Gas Chromatography

12.1N-nitrosodiphenylamine completely reacts to form diphenylamine at the normal operating temperatures of a GC injection port (200 to 250 °C). Thus, N-nitro-sodi-phenylamine is chromatographed and detected as di-phe-nyla-mine. Accurate determination depends on removal of di-phe-nyla-mine that may be present in the original extract prior to GC analysis (See Section 11).

12.2Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Column 1 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.3Calibrate the system daily as described in Section 7.

12.4If the extract has not been subjected to one of the cleanup procedures in Section 11, it is necessary to exchange the solvent from methylene chloride to methanol before the thermionic detector can be used. To a 1 to 10-mL volume of methylene chloride extract in a concentrator tube, add 2 mL of methanol and a clean boiling chip. Attach a two-ball micro-Snyder column to the concentrator tube. Prewet the column by adding about 0.5 mL of methylene chloride to the top. Place the micro-K-D apparatus on a boiling (100 °C) water bath so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with 0.2 mL of methanol. Adjust the final volume to 2.0 mL.

12.5If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

12.6Inject 2 to 5 μL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.21 Smaller (1.0 μL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 μL, and the resulting peak size in area or peak height units.

12.7Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.8If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.9If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations

13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

EC15NO91.105Equation 2where:A=Amount of material injected (ng).Vi=Volume of extract injected (μL).Vt=Volume of total extract (μL).Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

EC15NO91.106Equation 3where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Is=Amount of internal standard added to each extract (μg).Vo=Volume of water extracted (L).

13.2Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.3 The MDL concentrations listed in Table 1 were obtained using reagent water.22 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4 x MDL to 1000 x MDL.22

14.3This method was tested by 17 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 0.8 to 55 μg/L.23 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

5. ASTM Annual Book of Standards, Part 31, D3694-78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

17. “Scientific and Technical Assessment Report on Nitrosamines,” EPA-660/6-7-001, Office of Research and Development, U.S. Environmental Protection Agency (1976).

18. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value of 1.22 derived in this report.)

19. ASTM Annual Book of Standards, Part 31, D3370-76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

20. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA-600/4-79-020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

1.2This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 609, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1A measured volume of sample, approximately 1-L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the parameters are then measured with an electron capture detector.2

2.2The method provides a Florisil column cleanup procedure and an elemental sulfur removal procedure to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Interferences by phthalate esters can pose a major problem in pesticide analysis when using the electron capture detector. These compounds generally appear in the chromatogram as large late eluting peaks, especially in the 15 and 50% fractions from Florisil. Common flexible plastics contain varying amounts of phthalates. These phthalates are easily extracted or leached from such materials during laboratory operations. Cross contamination of clean glassware routinely occurs when plastics are handled during extraction steps, especially when solvent-wetted surfaces are handled. Interferences from phthalates can best be minimized by avoiding the use of plastics in the laboratory. Exhaustive cleanup of reagents and glassware may be required to eliminate background phthalate contamination.4, 5 The interferences from phthalate esters can be avoided by using a microcoulometric or electrolytic conductivity detector.

3.3Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedures in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

4. Safety

4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 6-8 for the information of the analyst.

4.2The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: 4,4′-DDT, 4,4′-DDD, the BHCs, and the PCBs. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials

5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle—1-L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during composting. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2.Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.4Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K-570050-1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

chart recorder. A data system is recommended for measuring peak areas.

5.6.1Column 1—1.8 m long × 4 mm ID glass, packed with 1.5% SP-2250/1.95% SP-2401 on Supelcoport (100/120 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.3Detector—Electron capture detector. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1.

6. Reagents

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.6.1Ethyl ether must be shown to be free of peroxides before it is used as indicated by EM Laboratories Quant test strips. (Available from Scientific Products Co., Cat. No. P1126-8, and other suppliers.)

6.6.2Procedures recommended for removal of peroxides are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.

6.8Florisil—PR grade (60/100 mesh). Purchase activated at 1250 °F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 130 °C in a foil-covered glass container and allow to cool.

6.11.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in isooctane and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.11.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.11.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.12Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure:

7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with isooctane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with isooctane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

EC15NO91.107Equation 1where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard (μg/L).Cs=Concentraton of the parameter to be measured (μg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, the test must be repeated using a fresh calibration standard. Alternatively, a new calibration curve must be prepared for that compound.

7.5The cleanup procedure in Section 11 utilizes Florisil column chromatography. Florisil from different batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil which is used, the use of lauric acid value 9 is suggested. The referenced procedure determines the adsorption from hexane solution of lauric acid (mg) per g of Florisil. The amount of Florisil to be used for each column is calculated by dividing 110 by this ratio and multiplying by 20 g.

7.6Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each single-component parameter of interest at the following concentrations in acetone: 4,4′-DDD, 10 μg/mL; 4,4′-DDT, 10 μg/mL; endosulfan II, 10 μg/mL; endosulfan sulfate, 10 μg/mL; endrin, 10 μg/mL; any other single-component pesticide, 2 μg/mL. If this method is only to be used to analyze for PCBs, chlordane, or toxaphene, the QC check sample concentrate should contain the most representative multicomponent parameter at a concentration of 50 μg/mL in acetone. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at the test concentrations shown in Table 3 by adding 1.00 mL of QC check sample concentrate to each of four 1-L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X) in μg/mL; and the standard deviation of the recovery (s) in μg/mL, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note:

The large number of parameters in Table 3 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.2.6.2Beginning with Section 8.2.2, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compmunds of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 3. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.10 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 3, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 4, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 4, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.10

8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory. If the entire list of parameters in Table 3 must be measured in the sample in Section 8.3, the probability that the analysis of a QC check standard will be required is high. In this case the QC check standard should be routinely analyzed with the spike sample.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standards to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 3. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P−2 sp to P+2 sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1Grab samples must be collected in glass containers. Conventional sampling practices 11 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 °C from the time of collection until extraction. If the samples will not be extracted within 72 h of collection, the sample should be adjusted to a pH range of 5.0 to 9.0 with sodium hydroxide solution or sulfuric acid. Record the volume of acid or base used. If aldrin is to be determined, add sodium thiosulfate when residual chlorine is present. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.12 Field test kits are available for this purpose.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel.

10.2Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optium technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.7Increase the temperature of the hot water bath to about 80 °C. Momeltarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Concentrate the extract as in Section 10.6, except use hexane to prewet the column. The elapsed time of concentration should be 5 to 10 min.

10.8Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chroma-tographic analysis (Section 12). If the sample -requires further cleanup, proceed to Sec-tion 11.

10.9Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use either procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure. The Florisil column allows for a select fractionation of the compounds and will eliminate polar interferences. Elemental sulfur, which interferes with the electron capture gas chromatography of certain pesticides, can be removed by the technique described in Section 11.3.

11.2Florisil column cleanup:

11.2.1Place a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.5), into a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm of anhydrous sodium sulfate to the top.

11.2.2Add 60 mL of hexane to wet and rinse the sodium sulfate and Florisil. Just prior to exposure of the sodium sulfate layer to the air, stop the elution of the hexane by closing the stopcock on the chromatographic column. Discard the eluate.

11.2.3Adjust the sample extract volume to 10 mL with hexane and transfer it from the K-D concentrator tube onto the column. Rinse the tube twice with 1 to 2 mL of hexane, adding each rinse to the column.

11.2.4Place a 500-mL K-D flask and clean concentrator tube under the chroma-tographic column. Drain the column into -the flask until the sodium sulfate layer -is nearly exposed. Elute the column with 200 mL of 6% ethyl ether in hexane (V/V) (Fraction 1) at a rate of about 5 mL/min. Remove the K-D flask and set it aside for later concentration. Elute the column again, using 200 mL of 15% ethyl ether in hexane (V/V) (Fraction 2), into a second K-D flask. Perform the third elution using 200 mL of 50% ethyl ether in hexane (V/V) (Fraction 3). The elution patterns for the pesticides and PCBs are shown in Table 2.

11.2.5Concentrate the fractions as in Section 10.6, except use hexane to prewet the column and set the water bath at about 85 °C. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with hexane. Adjust the volume of each fraction to 10 mL with hexane and analyze by gas chromatography (Section 12).

11.3Elemental sulfur will usually elute entirely in Fraction 1 of the Florisil column cleanup. To remove sulfur interference from this fraction or the original extract, pipet 1.00 mL of the concentrated extract into a clean concentrator tube or Teflon-sealed vial. Add one to three drops of mercury and seal.13 Agitate the contents of the vial for 15 to 30 s. Prolonged shaking (2 h) may be required. If so, this may be accomplished with a reciprocal shaker. Alternatively, activated copper powder may be used for sulfur removal.14 Analyze by gas chromatography.

12. Gas Chromatography

12.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Column 1 are shown in Figures 1 to 10. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2Calibrate the system daily as described in Section 7.

12.3If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

12.4Inject 2 to 5 μL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.15 Smaller (1.0 uL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 μL, the total extract volume, and the resulting peak size in area or peak height units.

12.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations

13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

EC15NO91.108Equation 2where:A=Amount of material injected (ng).Vi=Volume of extract injected (μL).Vt=Volume of total extract (μL).Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

EC15NO91.109Equation 3where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Is=Amount of internal standard added to each extract (μg).Vo=Volume of water extracted (L).

13.2When it is apparent that two or more PCB (Aroclor) mixtures are present, the Webb and McCall procedure 16 may be used to identify and quantify the Aroclors.

13.3For multicomponent mixtures (chlordane, toxaphene, and PCBs) match retention times of peaks in the standards with peaks in the sample. Quantitate every identifiable peak unless interference with individual peaks persist after cleanup. Add peak height or peak area of each identified peak in the chromatogram. Calculate as total response in the sample versus total response in the standard.

13.4Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.17 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4×MDL to 1000×MDL with the following exceptions: Chlordane recovery at 4×MDL was low (60%); Toxaphene recovery was demonstrated linear over the range of 10×MDL to 1000×MDL.17

14.3This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations.18 Concentrations used in the study ranged from 0.5 to 30 μg/L for single-component pesticides and from 8.5 to 400 μg/L for multicomponent parameters. Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 4.

3. ASTM Annual Book of Standards, Part 31, D3694-78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

9. Mills, P.A. “Variation of Florisil Activity: Simple Method for Measuring Absorbent Capacity and Its Use in Standardizing Florisil Columns,” Journal of the Association of Official Analytical Chemists, 51, 29, (1968).

10. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

11. ASTM Annual Book of Standards, Part 31, D3370-76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

12. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA-600/4-79-020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

14. “Manual of Analytical Methods for the Analysis of Pesticides in Human and Environmental Samples,” EPA-600/8-80-038, U.S. Environmental Protection Agency, Health Effects Research Laboratory, Research Triangle Park, North Carolina.

1.2This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1A measured volume of sample, approximately 1-L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. Isophorone and nitrobenzene are measured by flame ionization detector gas chromatography (FIDGC). The dinitrotoluenes are measured by electron capture detector gas chromatography (ECDGC).2

2.2The method provides a Florisil column cleanup procedure to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baseliles in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

4. Safety

4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 4‐6 for the information of the analyst.

5. Apparatus and Materials

5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle—1-L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.4Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K-570050-1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.4Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5Balance—Analytical, capable of accurately weighing 0.0001 g.

5.6Gas chromatograph—An analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1Column 1—1.2 m long x 2 or 4 mm ID glass, packed with 1.95% QF-1/1.5% OV-17 on Gas-Chrom Q (80/100 mesh) or equivalent. This column was used to develop the method performance statements given in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.3Detectors—Flame ionization and electron capture detectors. The flame ionization detector (FID) is used when determining isophorone and nitrobenzene. The electron capture detector (ECD) is used when determining the dinitrotoluenes. Both detectors have proven effective in the analysis of wastewaters and were used in develop the method performance statements in Section 14. Guidelines for the use to alternate detectors are provided in Section 12.1.

6. Reagents

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.7.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in hexane and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.8Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure:

7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with hexane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD) linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flash. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with hexane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

Equation 1.

EC15NO91.110where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard (μg/L).Cs=Concentration of the parameter to be measured (μg/L).

If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound.

7.5Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1,5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest in acetone at a concentration of 20 μg/mL for each dinitrotoluene and 100 μg/mL for isophorone and nitrobenzene. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at the test concentrations shown in Table 2 by adding 1.00 mL of QC check sample concentrate to each of four 1-L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determile background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100 (A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X8; (3) calculate the range for recovery at the spike concentration as (100 X′/T) ±2.44 (100 S′/T)%.7

8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4.If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P−2sp to P+2sp. If P=90% and sp = 10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1Grab samples must be collected in glass containers. Conventional sampling practices 8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 °C from the time of collection until extraction.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH of the sample with wide-range pH paper and adjust to within the range of 5 to 9 with sodium hydroxide solution or sulfuric acid.

10.2Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Sections 10.7 and 10.8 describe a procedure for exchanging the methylene chloride solvent to hexane while concentrating the extract volume to 1.0 mL. When it is not necessary to achieve the MDL in Table 2, the solvent exchange may be made by the addition of 50 mL of hexane and concentration to 10 mL as described in Method 606, Sections 10.7 and 10.8.

10.7Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.8Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Add 1 to 2 mL of hexane and a clean boiling chip to the concentrator tube and attach a two-ball micro-Snyder column. Prewet the column by adding about 0.5 mL of hexane to the top. Place the micro-K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.9Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of hexane. Adjust the extract volume to 1.0 mL. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.10Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2Florisil column cleanup:

11.2.1Prepare a slurry of 10 g of acti-vated Florisil in methylene chloride/-hexane (1+9)(V/V) and place the Florisil -into a chromatographic column. Tap the column to settle the Florisil and add 1 cm of anhydrous sodium sulfate to the top. Adjust the elution rate to about 2 mL/min.

11.2.2Just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the sample extract onto the column using an additional 2 mL of hexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 30 mL of methylene chloride/hexane (1 + 9)(V/V) and continue the elution of the column. Discard the eluate.

11.2.3Next, elute the column with 30 mL of acetone/methylene chloride (1 + 9)(V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction as in Sections 10.6, 10.7, 10.8, and 10.9 including the solvent exchange to 1 mL of hexane. This fraction should contain the nitroaromatics and isophorone. Analyze by gas chromatography (Section 12).

12. Gas Chromatography

12.1Isophorone and nitrobenzene are analyzed by injection of a portion of the extract into an FIDGC. The dinitrotoluenes are analyzed by a separate injection into an ECDGC. Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Column 1 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2Calibrate the system daily as described in Section 7.

12.3If the internal standard calibration procedure is being used, the internal standard must be added to the same extract and mixed thoroughly immediately before injection into the gas chromatograph.

12.4Inject 2 to 5 μL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.9 Smaller (1.0 μL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 μL, the total extract volume, and the resulting peak size in area or peak height units.

12.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations

13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

EC15NO91.111Equation 2where:A=Amount of material injected (ng).Vi=Volume of extract injected (μL).Vt=Volume of total extract (μL).Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

EC15NO91.112Equation 3where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Is=Amount of internal standard added to each extract (μg).Vo=Volume of water extracted (L).

13.2Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.10 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 7×MDL to 1000×MDL.10

14.3This method was tested by 18 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 515 μg/L.11 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

3. ASTM Annual Book of Standards, Part 31, D3694-78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

4. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77-206, August 1977.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

8. ASTM Annual Book of Standards, Part 31, D3370-76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

Table 1—Chromatographic Conditions and Method Detection LimitsParameterRetention time (min)Col. 1Col. 2Method detection limit (μg/L)ECDGCFIDGCNitrobenzene3.314.3113.73.62,6-Dinitrotoluene3.524.750.01−Isophorone4.495.7215.75.72,4-Dinitrotoluene5.356.540.02− AAColumn 1 conditions: Gas-Chrom Q (80/100 mesh) coated with 1.95% QF-1/1.5% OV-17 packed in a 1.2 m long × 2 mm or 4 mm ID glass column. A 2 mm ID column and nitrogen carrier gas at 44 mL/min flow rate were used when determining isophorone and nitrobenzene by FIDGC. The column temperature was held isothermal at 85 °C. A 4 mm ID column and 10% methane/90% argon carrier gas at 44 mL/min flow rate were used when determining the dinitrotol-uenes by ECDGC. The column temperature was held iso-thermal at 145 °C. AAColumn 2 conditions: Gas-Chrom Q (80/100 mesh) coated with 3% OV-101 packed in a 3.0 m long × 2 mm or 4 mm ID glass column. A 2 mm ID column and nitrogen carrier gas at 44 mL/min flow rate were used when determining isophorone and nitrobenzene by FIDGC. The column temperature was held isothermal at 100 °C. A 4 mm ID column and 10% methane/90% argon carrier gas at 44 mL/min flow rate were used when determining the dinitrotoluenes by ECDGC. The column temperature was held isothermal at 150 °C.Table 2—QC Acceptance Criteria—Method 609ParameterTest Conc. (μg/L)Limit for s (μg/L)Range for X (μg/L)Range for P, Ps (%)2,4-Dinitrotoluene205.13.6-22.86-1252,6-Dinitrotoluene204.83.8-23.08-126Isophorone10032.38.0-100.0D-117Nitrobenzene10033.325.7-100.06-118s=Standard deviation of four recovery measurements, in μg/L (Section 8.2.4).X=Average recovery for four recovery measurements, in μg/L (Section 8.2.4).P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).D=Detected; result must be greater than zero.Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.Table 3—Method Accuracy and Precision as Functions of Concentration—Method 609ParameterAccuracy, as recovery, X′ (μg/L)Single analyst precision, sr′ (μg/L)Overall precision, S′ (μg/L)2,4-Dinitro-toluene0.65C+0.220.20X+0.080.37X−0.072,6-Dinitro-toluene0.66C+0.200.19X+0.060.36X−0.00Isophorone0.49C+2.930.28X+2.770.46X+0.31Nitrobenzene0.60C+2.000.25X+2.530.37X−0.78X′=Expected recovery for one or more measurements of a sample containing a concentration of C, in μg/L.sr′=Expected single analyst standard deviation of measurements at an average concentration found of X, in μg/L.S′=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in μg/L.C=True value for the concentration, in μg/L.X=Average recovery found for measurements of samples containing a concentration of C, in μg/L.EC02JY92.029EC02JY92.030Method 610—Polynuclear Aromatic HydrocarbonsPt. 136, App. A, Meth. 6101. Scope and Application

1.1This method covers the determination of certain polynuclear aromatic hydrocarbons (PAH). The following parameters can be determined by this method:

1.2This is a chromatographic method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for many of the parameters listed above, using the extract produced by this method.

1.3This method provides for both high performance liquid chromatographic (HPLC) and gas chromatographic (GC) approaches for the determination of PAHs. The gas chromatographic procedure does not adequately resolve the following four pairs of compounds: Anthracene and phenanthrene; chrysene and benzo(a)anthracene; benzo(b)fluoranthene and benzo(k)fluoranthene; and dibenzo(a,h) anthracene and indeno (1,2,3-cd)pyrene. Unless the purpose for the analysis can be served by reporting the sum of an unresolved pair, the liquid chromatographic approach must be used for these compounds. The liquid chromatographic method does resolve all 16 of the PAHs listed.

1.4The method detection limit (MDL, defined in Section 15.1) 1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.5The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 609, 611, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. Selection of the aliquots must be made prior to the solvent exchange steps of this method. The analyst is allowed the latitude, under Sections 12 and 13, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.6Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.7This method is restricted to use by or under the supervision of analysts experienced in the use of HPLC and GC systems and in the interpretation of liquid and gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1A measured volume of sample, approximately 1-L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and concentrated to a volume of 10 mL or less. The extract is then separated by HPLC or GC. Ultraviolet (UV) and fluorescence detectors are used with HPLC to identify and measure the PAHs. A flame ionization detector is used with GC.2

2.2The method provides a silica gel column cleanup procedure to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardward that lead to discrete artifacts and/or elevated baselines in the chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

3.3The extent of interferences that may be encountered using liquid chromatographic techniques has not been fully assessed. Although the HPLC conditions described allow for a unique resolution of the specific PAH compounds covered by this method, other PAH compounds may interfere.

4. Safety

4.1The toxicity or carcinogenicity of each reagent used in this method have not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 4−6 for the information of the analyst.

4.2The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: benzo(a)anthracene, benzo(a)pyrene, and dibenzo(a,h)-anthracene. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials

5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle—1-L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.3Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K-570050-1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.6.3Detectors—Fluorescence and/or UV detectors. The fluorescence detector is used for excitation at 280 nm and emission greater than 389 nm cutoff (Corning 3-75 or equivalent). Fluorometers should have dispersive optics for excitation and can utilize either filter or dispersive optics at the emission detector. The UV detector is used at 254 nm and should be coupled to the fluorescence detector. These detectors were used to develop the method performance statements in Section 15. Guidelines for the use of alternate detectors are provided in Section 12.2.

5.7Gas chromatograph—An analytical system complete with temperature programmable gas chromatograph suitable for on-column or splitless injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.7.1Column—1.8 m long x 2 mm ID glass, packed with 3% OV-17 on Chromosorb W-AW-DCMS (100/120 mesh) or equivalent. This column was used to develop the retention time data in Table 2. Guidelines for the use of alternate column packings are provided in Section 13.3.

5.7.2Detector—Flame ionization detector. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), excluding the four pairs of unresolved compounds listed in Section 1.3. Guidelines for the use of alternate detectors are provided in Section 13.3.

6. Reagents

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.7.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in acetonitrile and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When com-pound purity is assayed to be 96% or greater, -the weight can be used without cor-rection to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.8Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1Establish liquid or gas chroma-tog-ra-phic operating conditions equivalent to those given in Table 1 or 2. The chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure:

7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with acetonitrile. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 5 to 25 μL for HPLC and 2 to 5 μL for GC, analyze each calibration standard according to Section 12 or 13, as appropriate. Tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with acetonitrile. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 5 to 25 μL for HPLC and 2 to 5 μL for GC, analyze each calibration standard according to Section 12 or 13, as appropriate. Tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

EC15NO91.113Equation 1where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard (μg/L).Cs=Concentration of the parameter to be measured (μg/L).If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, the test must be repeated using a fresh calibration standard. Alternatively, a new calibration curve must be prepared for that compound.

7.5Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, 12.2, and 13.3) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at the following concentrations in acetonitrile: 100 μg/mL of any of the six early-eluting PAHs (naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, and anthracene); 5 μg/mL of benzo(k)fluoranthene; and 10 μg/mL of any of the other PAHs. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at the test concentrations shown in Table 3 by adding 1.00 mL of QC check sample concentrate to each of four 1-L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 3. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note:

The large number of parameters in Table 3 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.2.6.2Beginning with Section 8.2.2, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none, (2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100 (A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 3. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 3, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 4, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 4, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.7

8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the critiera must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory. If the entire list of parameters in Table 3 must be measured in the sample in Section 8.3, the probability that the analysis of a QC check standard will be required is high. In this case the QC check standard should be routinely analyzed with the spike sample.

8.4.1Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 3. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P-2sp to P+2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1Grab samples must be collected in glass containers. Conventional sampling practices 8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 °C from the time of collection until extraction. PAHs are known to be light sensitive; therefore, samples, extracts, and standards should be stored in amber or foil-wrapped bottles in order to minimize photolytic decomposition. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.9 Field test kits are available for this purpose.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel.

10.2Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.7Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial and protected from light. If the sample extract requires no further cleanup, proceed with gas or liquid chromatographic analysis (Section 12 or 13). If the sample requires further cleanup, proceed to Section 11.

10.8Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the methods as revised to incorporate the cleanup procedure.

11.2Before the silica gel cleanup technique can be utilized, the extract solvent must be exchanged to cyclohexane. Add 1 to 10 mL of the sample extract (in methylene chloride) and a boiling chip to a clean K-D concentrator tube. Add 4 mL of cyclohexane and attach a two-ball micro-Snyder column. Prewet the column by adding 0.5 mL of methylene chloride to the top. Place the micro-K-D apparatus on a boiling (100 °C) water bath so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of the liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of cyclohexane. Adjust the extract volume to about 2 mL.

11.3Silica gel column cleanup for PAHs:

11.3.1Prepare a slurry of 10 g of activiated silica gel in methylene chloride and place this into a 10-mm ID chromatographic column. Tap the column to settle the silica gel and elute the methylene chloride. Add 1 to 2 cm of anhydrous sodium sulfate to the top of the silica gel.

11.3.2Preelute the column with 40 mL of pentane. The rate for all elutions should be about 2 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, transfer the 2-mL cyclohexane sample extract onto the column using an additional 2 mL cyclohexane to complete the transfer. Just prior to exposure of the sodium sulfate layer to the air, add 25 mL of pentane and continue the elution of the column. Discard this pentane eluate.

11.3.3Next, elute the column with 25 mL of methylene chloride/pentane (4+6)(V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction to less than 10 mL as in Section 10.6. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint with pentane. Proceed with HPLC or GC analysis.

12. High Performance Liquid Chromatography

12.1To the extract in the concentrator tube, add 4 mL of acetonitrile and a new boiling chip, then attach a two-ball micro-Snyder column. Concentrate the solvent as in Section 10.6, except set the water bath at 95 to 100 °C. When the apparatus is cool, remove the micro-Snyder column and rinse its lower joint into the concentrator tube with about 0.2 mL of acetonitrile. Adjust the extract volume to 1.0 mL.

12.2Table 1 summarizes the recommended operating conditions for the HPLC. Included in this table are retention times, capacity factors, and MDL that can be achieved under these conditions. The UV detector is recommended for the determination of naphthalene, acenaphthylene, acenapthene, and fluorene and the fluorescence detector is recommended for the remaining PAHs. Examples of the separations achieved by this HPLC column are shown in Figures 1 and 2. Other HPLC columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.3Calibrate the system daily as described in Section 7.

12.4If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the instrument.

12.5Inject 5 to 25 μL of the sample extract or standard into the HPLC using a high pressure syringe or a constant volume sample injection loop. Record the volume injected to the nearest 0.1 μL, and the resulting peak size in area or peak height units. Re-equilibrate the HPLC column at the initial gradient conditions for at least 10 min between injections.

12.6Identify the parameters in the sam-ple by comparing the retention time of -the peaks in the sample chromatogram with those of -the peaks in standard chromatograms. The -width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.7If the response for a peak exceeds the working range of the system, dilute the extract with acetonitrile and reanalyze.

12.8If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Gas Chromatography

13.1The packed column GC procedure will not resolve certain isomeric pairs as indicated in Section 1.3 and Table 2. The liquid chromatographic procedure (Section 12) must be used for these parameters.

13.2To achieve maximum sensitivity with this method, the extract must be concentrated to 1.0 mL. Add a clean boiling chip to the methylene chloride extract in the concentrator tube. Attach a two-ball micro-Snyder column. Prewet the micro-Snyder column by adding about 0.5 mL of methylene chloride to the top. Place the micro-K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with a minimum amount of methylene chloride. Adjust the final volume to 1.0 mL and stopper the concentrator tube.

13.3Table 2 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times that were obtained under these conditions. An example of the separations achieved by this column is shown in Figure 3. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

13.4Calibrate the gas chromatographic system daily as described in Section 7.

13.5If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatograph.

13.6Inject 2 to 5 μL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.10 Smaller (1.0 μL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 μL, and the resulting peak size in area or peak height units.

13.7Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

13.8If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

13.9If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

14. Calculations

14.1Determine the concentration of individual compounds in the sample.

14.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

EC15NO91.114Equation 2where:A=Amount of material injected (ng).Vi=Volume of extract injected (μL).Vt=Volume of total extract (μL).Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

EC15NO91.115Equation 3where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Is=Amount of internal standard added to each extract (μg).Vo=Volume of water extracted (L).

14.2Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

15. Method Performance

15.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.11 Similar results were achieved using representative wastewaters. MDL for the GC approach were not determined. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

15.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 8 × MDL to 800 × MDL11 with the following exception: benzo(ghi)perylene recovery at 80 × and 800 × MDL were low (35% and 45%, respectively).

15.3This method was tested by 16 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 0.1 to 425 μg/L.12 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 4.

3. ASTM Annual Book of Standards, Part 31, D3694-78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

4. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77-206, August 1977.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

8. ASTM Annual Book of Standards, Part 31, D3370-76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

9. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA-600/4-79-020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

1.2This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 609, and 612. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1A measured volume of sample, approximately 1-L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the parameters are then measured with a halide specific detector.2

2.2The method provides a Florisil column cleanup procedure to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.3 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed be detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such a PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

3.3Dichlorobenzenes are known to coelute with haloethers under some gas chromatographic conditions. If these materials are present together in a sample, it may be necessary to analyze the extract with two different column packings to completely resolve all of the compounds.

4. Safety

4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 4−6 for the information of the analyst.

5. Apparatus and Materials

5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle—1-L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.4Concentrator tube, Kuderna-Danish—-10-mL, graduated (Kontes K-570050-1025 or equival-ent). Calibration must be checked at the volumes em-ployed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.4Water bath—Heated, with concentric ring cover, capable of temperature control (±2°C). The bath should be used in a hood.

5.5Balance—Analytical, capable of accurately weighing 0.0001 g.

5.6Gas chromatograph—An analytical system complete with temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1Column 1—1.8 m long x 2 mm ID glass, -packed with 3% SP-1000 on Supel-co-port (100/120 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 12.1.

5.6.3Detector—Halide specific detector: electrolytic conductivity or microcoulo-metric. These detectors have proven effec-tive in the analysis of wastewaters for -the parameters listed in the scope (Section 1.1). The Hall conductivity detector was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1. Although less selective, an electron capture detector is an acceptable alternative.

6. Reagents

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.5Florisil—PR Grade (60/100 mesh). Purchase activated at 1250 °F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 130 °C in a foil-covered glass container and allow to cool.

6.6Ethyl ether—Nanograde, redistilled in glass if necessary.

6.6.1Ethyl ether must be shown to be free of peroxides before it is used as indicated by EM Laboratories Quant test strips. (Available from Scientific Products Co., Cat. No. P1126-8, and other suppliers.)

6.6.2Procedures recommended for removal of peroxides are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must be added to each liter of ether.

6.7.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in acetone and dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.7.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

6.8Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure:

7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with hexane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with hexane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

EC15NO91.116Equation 1where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard (μg/L).Cs=Concentration of the parameter to be measured (μg/L).If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound.

7.5The cleanup procedure in Section 11 utilizes Florisil column chromatography. Florisil from different batches or sources may vary in adsorptive capacity. To standardize the amount of Florisil which is used, the use of lauric acid value 7 is suggested. The referenced procedure determines the adsorption from hexane solution of lauric acid (mg) per g of Florisil. The amount of Florisil to be used for each column is calculated by dividing 110 by this ratio and multiplying by 20 g.

7.6Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate ac-cepta-ble accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 100 μg/mL in acetone. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at a concentration of 100 μg/L by adding 1.00 mL of QC check sample concentrate to each of four 1-L aliquots of reagent water.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter. Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1.The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 100 μg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or 100 μg/L.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.8 If spiking was performed at a concentration lower than 100 μg/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 S′/T)%.8

8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1Prepare the QC check standard by adding 1.0 m/L of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P-2sp to P+2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the -chromatogram, confirmatory techniques such as gas -chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1Grab samples must be collected in glass containers. Conventional sampling prac-tices9 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4°C from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.10 Field test kits are available for this purpose.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel.

10.2Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

Note:

Some of the haloethers are very volatile and significant losses will occur in concentration steps if care is not exercised. It is important to maintain a constant gentle evaporation rate and not to allow the liquid volume to fall below 1 to 2 mL before removing the K-D apparatus from the hot water bath.

10.7Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Raise the temperature of the water bath to 85 to 90 °C. Concentrate the extract as in Section 10.6, except use hexane to prewet the column. The elapsed time of concentration should be 5 to 10 min.

10.8Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw--cap vial. If the sample extract requires no -further cleanup, proceed with gas -chromatographic analysis (Section 12). If the sam-ple requires further cleanup, -proceed to Section 11.

10.9Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2Florisil column cleanup for haloethers:

11.2.1Adjust the sample extract volume to 10 mL.

11.2.2Place a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.5), into a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm of anhydrous sodium sulfate to the top.

11.2.3Preelute the column with 50 to 60 mL of petroleum ether. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the sample extract onto the column by decantation and subsequent petroleum ether washings. Discard the eluate. Just prior to exposure of the sodium sulfate layer to the air, begin eluting the column with 300 mL of ethyl ether/petroleum ether (6+94) (V/V). Adjust the elution rate to approximately 5 mL/min and collect the eluate in a 500-mL K-D flask equipped with a 10-mL concentrator tube. This fraction should contain all of the haloethers.

11.2.4Concentrate the fraction as in Section 10.6, except use hexane to prewet the column. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with hexane. Adjust the volume of the cleaned up extract to 10 mL with hexane and analyze by gas chromatography (Section 12).

12. Gas Chromatography

12.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Columns 1 and 2 are shown in Figures 1 and 2, respectively. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2Calibrate the system daily as described in Section 7.

12.3If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly immediately before injection into the gas chromatrograph.

12.4Inject 2 to 5 μL of the sample extract or standard into the gas chromatograph using the solvent-flush technique.11 Smaller (1.0 μL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 μL, the total extract volume, and the resulting peak size in area or peak height units.

12.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weight heavily in the interpretation of chromatograms.

12.6If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations

13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

EC15NO91.117Equation 2where:A=Amount of material injected (ng).Vi=Volume of extract injected (μL).Vt=Volume of total extract (μL).Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

EC15NO91.118Equation 3where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Is=Amount of internal standard added to each extract (μg).Vo=Volume of water extracted (L).

13.2Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.12 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4 × MDL to 1000 × MDL.12

14.3This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 626 μ/L.12 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

3. ASTM Annual Book of Standards, Part 31, D3694-78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constitutents,” American Society for Testing and Materials, Philadelphia.

7. Mills., P.A. “Variation of Florisil Activity: Simple Method for Measuring Absorbent Capacity and Its Use in Standardizing Florisil Columns,” Journal of the Association of Official Analytical Chemists, 51, 29 (1968).

8. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

9. ASTM Annual Book of Standards, Part 31, D3370-76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

10. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA-600/4-79-020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.

Table 1—Chromatographic Conditions and Methods Detection LimitsParametersRetention time (min)Column 1Column 2Method detection limit (μ/L)Bis(2-chloroisopropyl) ether8.49.70.8Bis(2-chloroethyl) ether9.39.10.3Bis(2-chloroethoxy) methane13.110.00.54-Chlorophenyl ether19.415.03.94-Bromophenyl phenyl ether21.216.22.3 AColumn 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-1000 packed in a 1.8 m long x 2 mm ID glass column with helium carrier gas at 40 mL/min. flow rate. Column temperature held at 60 °C for 2 min. after injection then programmed at 8 °C/min. to 230 °C and held for 4 min. Under these conditions the retention time for Aldrin is 22.6 min. AColumn 2 conditions: Tenax-GC (60/80 mesh) packed in a 1.8 m long x 2mm ID glass column with helium carrier gas at 40 mL/min. flow rate. Column temperature held at 150 °C for 4 min. after injection then programmed at 16 °C/min. to 310 °C. Under these conditions the retention time for Aldrin is 18.4 min.Table 2—QC Acceptance Criteria—Method 611ParameterTest conc. (μg/L)Limit for s (μg/L)Range for X (μg/L)Range for P, Ps percentBis (2-chloroethyl)ether10026.326.3-136.811-152Bis (2-chloroethoxy)methane10025.727.3-115.012-128Bis (2-chloroisopropyl)ether10032.726.4-147.09-1654-Bromophenyl phenyl ether10039.37.6 -167.5D-1894-Chlorophenyl phenyl ether10030.715.4-152.5D-170s=Standard deviation of four recovery measurements, in μg/L (Section 8.2.4).X=Average recovery for four recovery measurements, in μg/L (Section 8.2.4).P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).D=Detected; result must be greater than zero.Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.Table 3—Method Accuracy and Precision as Functions of Concentration—Method 611ParameterAccuracy, as recovery, X′ (μg/L)Single analyst precision, sr′ (μg/L)Overall precision, S′ (μg/L)Bis(2-chloroethyl) ether0.81C+0.540.19X+0.280.35X+0,36Bis(2-chloroethoxy) methane0.71C+0.130.20X+0.150.33X+0.11Bis(2-chloroisopropyl) ether0.85C+1.670.20X+1.050.36X+0.794-Bromophenyl phenyl ether0.85C+2.550.25X+0.210.47X+0.374-Chlorophenyl phenyl ether0.82C+1.970.18X+2.130.41X+0.55X′ = Expected recovery for one or more measuremelts of a sample containing a concentration of C, in μg/L.sr′ = Expected single analyst standard deviation of measurements at an average concentration found of X, in μg/L.S′ = Expected interlaboratory standard deviation of measurements at an average concentration found of X, in μg/L.C =True value for the concentration, in μg/L.X = Average recovery found for measurements of samples containing a concentration of C, in μg/L.EC02JY92.034EC02JY92.035Method 612—Chlorinated HydrocarbonsPt. 136, App. A, Meth. 6121. Scope and Application

1.1This method covers the determination of certain chlorinated hydrocarbons. The following parameters can be determined by this method:

1.2This is a gas chromatographic (GC) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for any or all of the compounds above, compound identifications should be supported by at least one additional qualitative technique. This method describes a second gas chromatographic column that can be used to confirm measurements made with the primary column. Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative and quantitative confirmation of results for all of the parameters listed above, using the extract produced by this method.

1.3The method detection limit (MDL, defined in Section 14.1)1for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.4The sample extraction and concentration steps in this method are essentially the same as in Methods 606, 608, 609, and 611. Thus, a single sample may be extracted to measure the parameters included in the scope of each of these methods. When cleanup is required, the concentration levels must be high enough to permit selecting aliquots, as necessary, to apply appropriate cleanup procedures. The analyst is allowed the latitude, under Section 12, to select chromatographic conditions appropriate for the simultaneous measurement of combinations of these parameters.

1.5Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph and in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1A measured volume of sample, approximately 1-L, is extracted with methylene chloride using a separatory funnel. The methylene chloride extract is dried and exchanged to hexane during concentration to a volume of 10 mL or less. The extract is separated by gas chromatography and the parameters are then measured with an electron capture detector.2

2.2The method provides a Florisil column cleanup procedure to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in gas chromatograms. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.3Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by this treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to minimize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. The cleanup procedure in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches to achieve the MDL listed in Table 1.

4. Safety

4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 4-6 for the information of the analyst.

5. Apparatus and Materials

5.1Sampling equipment, for discrete or composite sampling.

5.1.1Grab sample bottle—1cL or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.4Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K-570050-1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.4Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5Balance—Analytical, capable of accurately weighing 0.0001 g.

5.6Gas chromatograph—An analytical system complete with gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, gases, detector, and strip-chart recorder. A data system is recommended for measuring peak areas.

5.6.1Column 1—1.8 m long x 2 mm ID glass, packed with 1% SP-1000 on Supelcoport (100/120 mesh) or equivalent. Guidelines for the use of alternate column packings are provide in Section 12.1.

5.6.2Column 2—1.8 m long ×2 mm ID glass, packed with 1.5% OV-1/2.4% OV-225 on Supelcoport (80/100 mesh) or equivalent. This column was used to develop the method performance statements in Section 14.

5.6.3Detector— Electron capture detector. This detector has proven effective in the analysis of wastewaters for the parameters listed in the scope (Section 1.1), and was used to develop the method performance statements in Section 14. Guidelines for the use of alternate detectors are provided in Section 12.1.

6. Reagents

6.1Reagent water— Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.4Florisil—PR grade (60/100 mesh). Purchase activated at 1250 °F and store in the dark in glass containers with ground glass stoppers or foil-lined screw caps. Before use, activate each batch at least 16 h at 130 °C in a foil-covered glass container and allow to cool.

6.5.1Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material. Dissolve the material in isooctane and dilute to volume in a 120-mL volumetric flask. Larger volumes can be used at the convenience of the analyst. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.5.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at 4 °C and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.5.3Stock standard solutions must be replaced after six months, or sooner if comparision with check standards indicates a problem.

6.6Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1Establish gas chromatographic operating conditions equivalent to those given in Table 1. The gas chromatographic system can be calibrated using the external standard technique (Section 7.2) or the internal standard technique (Section 7.3).

7.2External standard calibration procedure:

7.2.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask and diluting to volume with isooctane. One of the external standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.2.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against the mass injected. The results can be used to prepare a calibration curve for each compound. Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over the working range (<10% relative standard deviation, RSD), linearity through the origin can be assumed and the average ratio or calibration factor can be used in place of a calibration curve.

7.3Internal standard calibration procedure—To use this approach, the analyst must select one or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Because of these limitations, no internal standard can be suggested that is applicable to all samples.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter of interest by adding volumes of one or more stock standards to a volumetric flask. To each calibration standard, add a known constant amount of one or more internal standards, and dilute to volume with isooctane. One of the standards should be at a concentration near, but above, the MDL and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the detector.

7.3.2Using injections of 2 to 5 μL, analyze each calibration standard according to Section 12 and tabulate peak height or area responses against concentration for each compound and internal standard. Calculate response factors (RF) for each compound using Equation 1.

EC15NO91.119Equation 1where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Cis=Concentration of the internal standard (μg/L).Cs=Concentration of the parameter to be measured (μg/L).If the RF value over the working range is a constant (<10% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4The working calibration curve, calibration factor, or RF must be verified on each working day by the measurement of one or more calibration standards. If the response for any parameter varies from the predicted response by more than ±15%, a new calibration curve must be prepared for that compound.

7.5Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When the results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at the following concentrations in acetone: Hexachloro-substituted parameters, 10 μg/mL; any other chlorinated hydrocarbon, 100 μg/mL. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at the test concentrations shown in Table 2 by adding 1.00 mL of QC check sample concentrate to each of four 1-L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for each parameter using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note:

The large number of parameters in Table 2 presents a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.2.

8.2.6.2Beginning with Section 8.2.2, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spike sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at the test concentration in Section 8.2.2 or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none by (2) the larger of either 5 times higher than the expected background concentration or the test concentration in Section 8.2.2.

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of each parameter. In necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100 (A-B)%/T, where T is the known true value of the spike.

8.3.3 Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7If spiking was performed at a concentration lower than the test concentration in Section 8.2.2, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of a parameter: (1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T) ± 2.44 (100 S′/T)%.7

8.3.4 If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4. If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory.

8.4.1 Prepare the QC check standard by adding 1.0 mL of QC check sample concentrate (Sections 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) for each parameter with the corresponding QC acceptance criteria found in Table 2. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P−2sp to P+2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. When doubt exists over the identification of a peak on the chromatogram, confirmatory techniques such as gas chromatography with a dissimilar column, specific element detector, or mass spectrometer must be used. Whenever possible, the laboratory should analyze standard reference materials and participate in relevent performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1Grab samples must be collected in glass containers. Conventional sampling practices 8 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 °C from the time of collection until extraction.

9.3All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel.

10.2Add 60 mL of methylele chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the volume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.3Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.4Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.5Pour the combined extract through a solvent-rinsed drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.

10.6Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 to 2 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

Note:

The dichloribenzenes have a sufficiently high volatility that significant losses may occur in concentration steps if care is not exercised. It is important to maintain a constant gentle evaporation rate and not to allow the liquid volume to fall below 1 to 2 mL before removing the K-D apparatus from the hot water bath.

10.7Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Raise the tempeature of the water bath to 85 to 90 °C. Concentrate the extract as in Section 10.6, except use hexane to prewet the column. The elapsed time of concentration should be 5 to 10 min.

10.8Romove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Stopper the concentrator tube and store refrigerated if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with gas chromatographic analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.9Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use the procedure below or any other appropriate procedure. However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure.

11.2Florisil column cleanup for chlorinated hydrocarbons:

11.2.1Adjust the sample extract to 10 mL with hexane.

11.2.2Place 12 g of Florisil into a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm of anhydrous sodium sulfate to the top.

11.2.3Preelute the column with 100 mL of petroleum ether. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the sample extract onto the column by decantation and subsequent petroleum ether washings. Discard the eluate. Just prior to exposure of the sodium sulfate layer to the air, begin eluting the column with 200 mL of petroleum ether and collect the eluate in a 500-mL K-D flask equipped with a 10-mL concentrator tube. This fraction should contain all of the chlorinated hydrocarbons.

11.2.4Concentrate the fraction as in Section 10.6, except use hexane to prewet the column. When the apparatus is cool, remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with hexane. Analyze by gas chromatography (Section 12).

12. Gas Chromatography

12.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Examples of the separations achieved by Columl 2 are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns, chromatographic conditions, or detectors may be used if the requirements of Section 8.2 are met.

12.2Calibrate the system daily as described in Section 7.

12.3If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed throughly immediately before injection into the gas chromatograph.

12.4Inject 2 to 5 μL of the sample extract or standard into the gas chromatograph using the solvent-flush techlique.9 Smaller (1.0 μL) volumes may be injected if automatic devices are employed. Record the volume injected to the nearest 0.05 μL, the total extract volume, and the resulting peak size in area or peak height units.

12.5Identify the parameters in the sample by comparing the retention times of the peaks in the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day. Three times the standard deviation of a retention time for a compound can be used to calculate a suggested window size; however, the experience of the analyst should weigh heavily in the interpretation of chromatograms.

12.6If the response for a peak exceeds the working range of the system, dilute the extract and reanalyze.

12.7If the measurement of the peak response is prevented by the presence of interferences, further cleanup is required.

13. Calculations

13.1Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of material injected from the peak response using the calibration curve or calibration factor determined in Section 7.2.2. The concentration in the sample can be calculated from Equation 2.

EC15NO91.120Equation 2where:A=Amount of material injected (ng).Vi=Volume of extract injected (μL).Vt=Volume of total extract (μL).Vs=Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.2 and Equation 3.

EC15NO91.121Equation 3where:As=Response for the parameter to be measured.Ais=Response for the internal standard.Is=Amount of internal standard added to each extract (μg).Vo=Volume of water extracted (L).

13.2Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.10 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method has been tested for linearity of spike recovery from reagent water and has been demonstrated to be applicable over the concentration range from 4×MDL to 1000×MDL.10

14.3This method was tested by 20 laboratories using reagent water, drinking water, surface water, and three industrial wastewaters spiked at six concentrations over the range 1.0 to 356 μg/L.11 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

3. ASTM Annual Book of Standards, Part 31, D3694-78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

4. “Carcinogens—Working With Carcinogens,” Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Publication No. 77-206, August 1977.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,”American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22 derived in this report.)

8. ASTM Annual Book of Standards, Part 31, D3370-76. “Standard Practices for Sampling Water,” American Society for Testing and Materials, Philadelphia.

Table 1—Chromatographic Conditions and Method Detection LimitsParameterRetention time (min)Column 1Column 2Method detection limit (μg/L)1,3-Dichlorobenzene4.56.81.19Hexachloroethane4.98.30.031,4-Dichlorobenzene5.27.61.341,2-Dichlorobenzene6.69.31.14Hexachlorobutadiene7.720.00.341,2,4-Trichlorobenzene15.522.30.05Hexachlorocyclopentadienendc 16.50.402-Chloronaphthalenea 2.7b 3.60.94Hexachlorobenzenea 5.6b 10.10.05Column 1 conditions: Supelcoport (100/120 mesh) coated with 1% SP-1000 packed in a 1.8 m x 2 mm ID glass column with 5% methane/95% argon carrier gas at 25 mL/min. flow rate. Column temperature held isothermal at 65 °C, except where otherwise indicated.Column 2 conditions: Supelcoport (80/100 mesh) coated with 1.5% OV-1/2.4% OV-225 packed in a 1.8 m x 2 mm ID glass column with 5% methane/95% argon carrier gas at 25 mL/min. flow rate. Column temperature held isothermal at 75 °C, except where otherwise indicated.nd=Not determined.a 150 °C column temperature.b 165 °C column temperature.c 100 °C column temperature.Table 2—QC Acceptance Criteria—Method 612ParameterTest conc. (μg/L)Limit for s (μg/L)Range for X (μg/L)Range for P, Ps (percent)2-Chloronaphthalene10037.329.5-126.99-1481,2-Dichlorobenzene10028.323.5-145.19-1601,3-Dichlorobenzene10026.47.2-138.6D-1501,4-Dichlorobenzene10020.822.7-126.913-137Hexachlorobenzene102.42.6-14.815-159Hexachlorobutadiene102.2D-12.7D-139Hexachlorocyclopentadiene102.5D-10.4D-111Hexachloroethane103.32.4-12.38-1391,2,4-Trichlorobenzene10031.620.2-133.75-149s=Standard deviation of four recovery measurements, in μg/L (Section 8.2.4).X=Average recovery for four recovery measurements, in μg/L (Section 8.2.4).P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).D=Detected; result must be greater than zero.Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.Table 3—Method Accuracy and Precision as Functions of Concentration—Method 612ParameterAcccuracy, as recovery, X′ (μg/L)Single analyst precision, sr′ (μg/L)Overall precision, S′ (μg/L)2-Chloronaphthalene0.75C+3.210.28X−1.170.38X−1.391,2-Dichlorobenzene0.85C−0.700.22X−2.950.41X−3.921,3-Dichlorobenzene0.72C+0.870.21X−1.030.49X−3.981,4-Dichlorobenzene0.72C+2.800.16X−0.480.35X−0.57Hexachlorobenzene0.87C−0.020.14X+0.070.36X−0.19Hexachlorobutadiene0.61C+0.030.18X+0.080.53X−0.12Hexachlorocyclopentadiene a0.47C0.24X0.50XHexachloroethane0.74C−0.020.23X+0.070.36X−0.001,2,4-Trichlorobenzene0.76C+0.980.23X−0.440.40X−1.37X′=Expected recovery for one or more measurements of a sample containing a concentration of C, in μg/L.sr′=Expected single analyst standard deviation of measurements at an average concentration found of X, in μg/L.S′=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in μg/L.C=True value for the concentration, in μg/L.X=Average recovery found for measurements of samples containing a concentration of C, in μg/L.a Estimates based upon the performance in a single laboratory.12EC02JY92.036EC02JY92.037Method 613—2,3,7,8-Tetrachlorodibenzo-p-DioxinPt. 136, App. A, Meth. 6131. Scope and Application

1.1This method covers the determination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The following parameter may be determined by this method:

ParameterSTORET No.GAS No.2,3,7,8-TCDD346751746-01-6

1.2This is a gas chromatographic/mass spectrometer (GC/MS) method applicable to the determination of 2,3,7,8-TCDD in municipal and industrial discharges as provided under 40 CFR 136.1. Method 625 may be used to screen samples for 2,3,7,8-TCDD. When the screening test is positive, the final qualitative confirmation and quantification must be made using Method 613.

1.3The method detection limit (MDL, defined in Section 14.1) 1 for 2,3,7,8-TCDD is listed in Table 1. The MDL for a specific wastewater may be different from that listed, depending upon the nature of interferences in the sample matrix.

1.4Because of the extreme toxicity of this compound, the analyst must prevent exposure to himself, of to others, by materials knows or believed to contain 2,3,7,8-TCDD. Section 4 of this method contains guidelines and protocols that serve as minimum safe-handling standards in a limited-access laboratory.

1.5Any modification of this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5.

1.6This method is restricted to use by or under the supervision of analysts experienced in the use of a gas chromatograph/mass spectrometer and in the interpretation of mass spectra. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1A measured volume of sample, approximately 1-L, is spiked with an internal standard of labeled 2,3,7,8-TCDD and extracted with methylene chloride using a separatory funnel. The methylene chloride extract is exchanged to hexane during concentration to a volume of 1.0 mL or less. The extract is then analyzed by capillary column GC/MS to separate and measure 2,3,7,8-TCDD.2,3

2.2The method provides selected column chromatographic cleanup proceudres to aid in the elimination of interferences that may be encountered.

3. Interferences

3.1Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample processing hardware that lead to discrete artifacts and/or elevated backgrounds at the masses (m/z) monitored. All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3.

3.1.1Glassware must be scrupulously cleaned.4 Clean all glassware as soon as possible after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent washing with hot water, and rinses with tap water and distilled water. The glassware should then be drained dry, and heated in a muffle furnace at 400 °C for 15 to 30 min. Some thermally stable materials, such as PCBs, may not be eliminated by the treatment. Solvent rinses with acetone and pesticide quality hexane may be substituted for the muffle furnace heating. Thorough rinsing with such solvents usually eliminates PCB interference. Volumetric ware should not be heated in a muffle furnace. After drying and cooling, glassware should be sealed and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store inverted or capped with aluminum foil.

3.1.2The use of high purity reagents and solvents helps to mininmize interference problems. Purification of solvents by distillation in all-glass systems may be required.

3.2Matrix interferences may be caused by contaminants that are coextracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature and diversity of the industrial complex or municipality being sampled. 2,3,7,8-TCDD is often associated with other interfering chlorinated compounds which are at concentrations several magnitudes higher than that of 2,3,7,8-TCDD. The cleanup producers in Section 11 can be used to overcome many of these interferences, but unique samples may require additional cleanup approaches 1,5-7 to eliminate false positives and achieve the MDL listed in Table 1.

3.3The primary column, SP-2330 or equivalent, resolves 2,3,7,8-TCDD from the other 21 TCDD insomers. Positive results using any other gas chromatographic column must be confirmed using the primary column.

4. Safety

4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified 8-10 for the information of the analyst. Benzene and 2,3,7,8-TCDD have been identified as suspected human or mammalian carcinogens.

4.2Each laboratory must develop a strict safety program for handling 2,3,7,8-TCDD. The following laboratory practices are recommended:

4.2.1Contamination of the laboratory will be minimized by conducting all manipulations in a hood.

4.2.2The effluents of sample splitters for the gas chromatograph and roughing pumps on the GC/MS should pass through either a column of activated charcoal or be bubbled through a trap containing oil or high-boiling alcohols.

4.2.3Liquid waste should be dissolved in methanol or ethanol and irradiated with ultraviolet light with a wavelength greater than 290 nm for several days. (Use F 40 BL lamps or equivalent). Analyze liquid wastes and dispose of the solutions when 2,3,7,8-TCDD can no longer be detected.

4.3Dow Chemical U.S.A. has issued the following precautimns (revised November 1978) for safe handling of 2,3,7,8-TCDD in the laboratory:

4.3.1The following statements on safe handling are as complete as possible on the basis of available toxicological information. The precautions for safe handling and use are necessarily general in nature since detailed, specific recommendations can be made only for the particular exposure and circumstances of each individual use. Inquiries about specific operations or uses may be addressed to the Dow Chemical Company. Assistance in evaluating the health hazards of particular plant conditions may be obtained from certain consulting laboratories and from State Departments of Health or of Labor, many of which have an industrial health service. 2,3,7,8-TCDD is extremely toxic to laboratory animals. However, it has been handled for years without injury in analytical and biological laboratories. Techniques used in handling radioactive and infectious materials are applicable to 2,3,7,8,-TCDD.

4.3.1.5Waste—Good technique includes mini-miz-ing contaminated waste. Plastic bag liners should be used in waste cans. Janitors must be trained in the safe handling of waste.

4.3.1.6Disposal of wastes—2,3,7,8-TCDD decomposes above 800 °C. Low-level waste such as absorbent paper, tissues, animal remains, and plastic gloves may be burned in a good incinerator. Gross quantities (milligrams) should be packaged securely and disposed through commercial or governmental channels which are capable of handling high-level radioactive wastes or extremely toxic wastes. Liquids should be allowed to evaporate in a good hood and in a disposable container. Residues may then be handled as above.

4.3.1.7Decontamination—For personal decontamination, use any mild soap with plenty of scrubbing action. For decontamination of glassware, tools, and surfaces, Chlorothene NU Solvent (Trademark of the Dow Chemical Company) is the least toxic solvent shown to be effective. Satisfactory cleaning may be accomplished by rinsing with Chlorothene, then washing with any detergent and water. Dishwater may be disposed to the sewer. It is prudent to minimize solvent wastes because they may require special disposal through commercial sources which are expensive.

4.3.1.8Laundry—Clothing known to be contaminated should be disposed with the precautions described under Section 4.3.1.6. Lab coats or other clothing worn in 2,3,7,8-TCDD work areas may be laundered.

Clothing should be collected in plastic bags. Persons who convey the bags and launder the clothing should be advised of the hazard and trained in proper handling. The clothing may be put into a washer without contact if the launderer knows the problem. The washer should be run through a cycle before being used again for other clothing.

4.3.1.9Wipe tests—A useful method of determining cleanliness of work surfaces and tools is to wipe the surface with a piece of filter paper. Extraction and analysis by gas chromatography can achieve a limit of sensitivity of 0.1 μg per wipe. Less than 1 μg of 2,3,7,8-TCDD per sample indicates acceptable cleanliness; anything higher warrants further cleaning. More than 10 μg on a wipe sample constitutes an acute hazard and requires prompt cleaning before further use of the equipment or work space. A high (>10 μg) 2,3,7,8-TCDD level indicates that unacceptable work practices have been employed in the past.

4.3.1.10Inhalation—Any procedure that may produce airborne contamination must be done with good ventilation. Gross losses to a ventilation system must not be allowed. Handling of the dilute solutions normally used in analytical and animal work presents no inhalation hazards except in the case of an accident.

5.1.1Grab sample bottle—1-L or 1-qt, amber glass, fitted with a screw cap lined with Teflon. Foil may be substituted for Teflon if the sample is not corrosive. If amber bottles are not available, protect samples from light. The bottle and cap liner must be washed, rinsed with acetone or methylene chloride, and dried before use to minimize contamination.

5.1.2Automatic sampler (optional)—The sampler must incorporate glass sample containers for the collection of a minimum of 250 mL of sample. Sample containers must be kept refrigerated at 4 °C and protected from light during compositing. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used. Before use, however, the compressible tubing should be thoroughly rinsed with methanol, followed by repeated rinsings with distilled water to minimize the potential for contamination of the sample. An integrating flow meter is required to collect flow proportional composites.

5.1.3Clearly label all samples as “POISON” and ship according to U.S. Department of Transportation regulations.

5.2Glassware (All specifications are suggested. Catalog numbers are included for illustration only.):

5.2.1Separatory funnels—2-L and 125-mL, with Teflon stopcock.

5.2.2Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes K-570050-1025 or equivalent). Calibration must be checked at the volumes employed in the test. Ground glass stopper is used to prevent evaporation of extracts.

5.4Water bath—Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath should be used in a hood.

5.5GC/MS system:

5.5.1Gas chromatograph—An analytical system complete with a temperature programmable gas chromatograph and all required accessories including syringes, analytical columns, and gases. The injection port must be designed for capillary columns. Either split, splitless, or on-column injection techniques may be employed, as long as the requirements of Section 7.1.1 are achieved.

5.5.2Column—60 m long × 0.25 mm ID glass or fused silica, coated with SP-2330 (or equivalent) with a film thickness of 0.2 μm. Any equivalent column must resolve 2, 3, 7, 8-TCDD from the other 21 TCDD isomers.16

5.5.3Mass spectrometer—Either a low resolution mass spectrometer (LRMS) or a high resolution mass spectrometer (HRMS) may be used. The mass spectrometer must be equipped with a 70 V (nominal) ion source and be capable of aquiring m/z abundance data in real time selected ion monitoring (SIM) for groups of four or more masses.

5.5.4GC/MS interface—Any GC to MS interface can be used that achieves the requirements of Section 7.1.1. GC to MS interfaces constructed of all glass or glass-lined materials are recommended. Glass surfaces can be deactivated by silanizing with dichlorodimethylsilane. To achieve maximum sensitivity, the exit end of the capillary column should be placed in the ion source. A short piece of fused silica capillary can be used as the interface to overcome problems associated with straightening the exit end of glass capillary columns.

5.5.5The SIM data acquired during the chromatographic program is defined as the Selected Ion Current Profile (SICP). The SICP can be acquired under computer control or as a real time analog output. If computer control is used, there must be software available to plot the SICP and report peak height or area data for any m/z in the SICP between specified time or scan number limits.

5.6Balance—Analytical, capable of accurately weighing 0.0001 g.

6. Reagents

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of 2, 3, 7, 8-TCDD.

6.2Sodium hydroxide solution (10 N)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute to 100 mL. Wash the solution with methylene chloride and hexane before use.

6.9Stock standard solutions (1.00 μg/μL)—Stock standard solutimns can be prepared from pure standard materials or purchased as certified solutions. Acetone should be used as the solvent for spiking solutions; ortho-xylene is recommended for calibration standards for split injectors; and tetradecane is recommended for splitless or on-colum injectors. Analyze stock internal standards to verify the absence of native 2,3,7,8-TCDD.

6.9.1Prepare stock standard solutions of 2,3,7,8-TCDD (mol wt 320) and either 37C14 2,3,7,8-TCDD (mol wt 328) or 13C112 2,3,7,8-TCDD (mol wt 332) in an isolated area by accurately weighing about 0.0100 g of pure material. Dissolve the material in pesticide quality solvent and dilute to volume in a 10-mL volumetric flask. When compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards can be used at any concentration if they are certified by the manufacturer or by an independent source.

6.9.2Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store in an isolated refrigerator protected from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards or spiking solutions from them.

6.9.3Stock standard solutions must be replaced after six months, or sooner if comparison with check standards indicates a problem.

7.1Establish gas chromatograhic operating conditions equivalent to those given in Table 1 and SIM conditions for the mass spectrometer as described in Section 12.2 The GC/MS system must be calibrated using the internal standard technique.

7.1.1Using stock standards, prepare calibration standards that will allow measurement of relative response factors of at least three concentration ratios of 2,3,7,8-TCDD to internal standard. Each calibration standard must be prepared to contain the internal standard at a concentration of 25 ng/mL. If any interferences are contributed by the internal standard at m/z 320 and 322, its concentration may be reduced in the calibration standards and in the internal standard spiking solution (Section 6.10). One of the calibration standards should contain 2,3,7,8-TCDD at a concentration near, but above, the MDL and the other 2,3,7,8-TCDD concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the GC/MS system.

7.1.2Using injections of 2 to 5 μL, analyze each calibration standardaccording to Section 12 and tabulate peak height or area response against the concentration of 2,3,7,8-TCDD and internal standard. Calculate response factors (RF) for 2,3,7,8-TCDD using Equation 1.

EC15NO91.122Equation 1where:As=SIM response for 2,3,7,8-TCDD m/z 320.Ais=SIM response for the internal standard, m/z 332 for 13 C12 2,3,7,8-TCDD m/z 328 for 37 Cl4 2,3,7,8-TCDD.Cis=Concentration of the internal standard (μg/L).Cs=Concentration of 2,3,7,8-TCDD (μg/L).If the RF value over the working range is a constant (<10% relative standard deviation, RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.1.3The working calibration curve or RF must be verified on each working day by the measurement of one or more 2,3,7,8-TCDD calibration standards. If the response for 2,3,7,8-TCDD varies from the predicted response by more than ±15%, the test must be repeated using a fresh calibration standard. Alternatively, a new calibration curve must be prepared.

7.2Before using any cleanup procedure, the analyst must process a series of calibration standards through the procedure to validate elution patterns and the absence of interferences from the reagents.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Sections 10.5, 11.1, and 12.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2

8.1.3Before processing any samples, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system and glassware are under control. Each time a set of samples is extracted or reagents are changed, a reagent water blank must be processed as a safeguard against laboratory contamination.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 10% of all samples with native 2,3,7,8-TCDD to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 10% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.5.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing 2,3,7,8-TCDD at a concentration of 0.100 μg/mL in acetone. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.2Using a pipet, prepare QC check samples at a concentration of 0.100 μg/L (100 ng/L) by adding 1.00 mL of QC check sample concentrate to each of four 1-L aliquots of reagent water.

8.2.3Analyze the well-mixed QC check samples according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for 2,3,7,8-TCDD using the four results.

8.2.5Compare s and (X) with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 2. If s and X meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If s exceeds the precision limit or X falls outside the range for accuracy, the system performance is unacceptable for 2,3,7,8-TCDD. Locate and correct the source of the problem and repeat the test beginning with Section 8.2.2.

8.3The laboratory must, on an ongoing basis, spike at least 10% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing one to ten samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of 2,3,7,8-TCDD in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of 2,3,7,8-TCDD in the sample is not being checked against a limit specific to that parameter, the spike should be at 0.100 μg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g., maximum holding times will be exceeded), the spike concentration should be (1) the regulatory concentration limit, if any; or, if none (2) the larger of either 5 times higher than the expected background concentration or 0.100 μg/L.

8.3.2Analyze one sample aliquot to determine the background concentration (B) of 2,3,7,8-TCDD. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentration in the sample. Spike a second sample aliquot with 1.0 mL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of 2,3,7,8-TCDD. Calculate percent recovery (P) as 100(A-B)%T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for 2,3,7,8-TCDD with the corresponding QC acceptance criteria found in Table 2. These acceptance criteria were calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.11 If spiking was performed at a concentration lower than 0.100 μg/L, the analyst must use either the QC acceptance criteria in Table 2, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recovery of 2,3,7,8-TCDD: (1) Calculate accuracy (X′) using the equation in Table 3, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 3, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T)±2.44(100 -S′/T)%. 11

8.3.4If the recovery of 2,3,7,8-TCDD falls outside the designated range for recovery, a check standard must be analyzed as described in Section 8.4.

8.4If the recovery of 2,3,7,8-TCDD fails the acceptance criteria for recovery in Section 8.3, a QC check standard must be prepared and analyzed.

Note:

The frequency for the required analysis of a QC check standard will depend upon the complexity of the sample matrix and the performance of the laboratory.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of 2,3,7,8-TCDD. Calculate the percent recovery (Ps) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (Ps) with the corresponding QC acceptance criteria found in Table 2. If the recovery of 2,3,7,8-TCDD falls outside the designated range, the laboratory performance is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for 2,3,7,8-TCDD in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the spandard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P−2sp to P+2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment on a regular basis (e.g. after each five to ten new accuracy measurements).

8.6It is recommended that the lab-o-ra-tory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1Grab samples must be collected in glass containers. Conventional sampling practices 12 should be followed, except that the bottle must not be prerinsed with sample before collection. Composite samples should be collected in refrigerated glass containers in accordance with the requirements of the program. Automatic sampling equipment must be as free as possible of Tygon tubing and other potential sources of contamination.

9.2All samples must be iced or refrigerated at 4 °C and protected from light from the time of collection until extraction. Fill the sample bottles and, if residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.13 Field test kits are available for this purpose.

9.3Label all samples and containers “POISON” and ship according to applicable U.S. Department of Transportation regulations.

9.4All samples must be extracted within 7 days of collection and completely analyzed within 40 days of extraction.2

10. Sample Extraction

Caution: When using this method to analyze for 2,3,7,8-TCDD, all of the following operations must be performed in a limited-access laboratory with the analyst wearing full protective covering for all exposed skin surfaces. See Section 4.2.

10.1Mark the water meniscus on the side of the sample bottle for later determination of sample volume. Pour the entire sample into a 2-L separatory funnel.

10.2Add 1.00 mL of internal standard spiking solution to the sample in the separatory funnel. If the final extract will be concentrated to a fixed volume below 1.00 mL (Section 12.3), only that volume of spiking solution should be added to the sample so that the final extract will contain 25 ng/mL of internal standard at the time of analysis.

10.3Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 s to rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample by shaking the funnel for 2 min. with periodic venting to release excess pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min. If the emulsion interface between layers is more than one-third the vmlume of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.

10.4Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extraction procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction in the same manner.

10.5Assemble a Kuderna-Danish (K-D) con-cen-tra-tor by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other concentration devices or techniques may be used in place of the K-D concentrator if the requirements of Section 8.2 are met.

10.6Pour the combined extract into the K-D concentrator. Rinse the Erlenmeyer flask with 20 to 30 mL of methylele chloride to complete the quantitative transfer.

10.7Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath (60 to 65 °C) so that the concentrator tube is partially immersed in the hot water, and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15 to 20 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood with condensed solvent. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min.

10.8Momentarily remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reattach the Snyder column. Raise the temperature of the water bath to 85 to 90°C. Concentrate the extract as in Section 10.7, except use hexane to prewet the column. Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. Set aside the K-D glassware for reuse in Section 10.14.

10.9Pour the hexane extract from the concentrator tube into a 125-mL separatory funnel. Rinse the concentrator tube four times with 10-mL aliquots of hexane. Combine all rinses in the 125-mL separatory funnel.

10.10Add 50 mL of sodium hydroxide solution to the funnel and shake for 30 to 60 s. Discard the aqueous phase.

10.11Perform a second wash of the organic layer with 50 mL of reagent water. Discard the aqueous phase.

10.12Wash the hexane layer with a least two 50-mL aliquots of concentrated sulfuric acid. Continue washing the hexane layer with 50-mL aliquots of concentrated sulfuric acid until the acid layer remains colorless. Discard all acid fractions.

10.13Wash the hexane layer with two 50-mL aliquots of reagent water. Discard the aqueous phases.

10.14Transfer the hexane extract into a 125-mL Erlenmeyer flask containing 1 to 2 g of anhydrous sodium sulfate. Swirl the flask for 30 s and decant the hexane extract into the reassembled K-D apparatus. Complete the quantitative transfer with two 10-mL hexane rinses of the Erlenmeyer flask.

10.15Replace the one or two clean boiling chips and concentrate the extract to 6 to 10 mL as in Section 10.8.

10.16Add a clean boiling chip to the concentrator tube and attach a two-ball micro-Snyder column. Prewet the column by adding about 1 mL of hexane to the top. Place the micro-K-D apparatus on the water bath so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 5 to 10 min. At the proper rate of distillation the balls of the column will actively chatter but the chambers will not flood. When the apparent volume of liquid reaches about 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min. Remove the micro-Snyder column and rinse its lower joint into the concentrator tube with 0.2 mL of hexane.

Adjust the extract volume to 1.0 mL with hexane. Stopper the concentrator tube and store refrigerated and protected from light if further processing will not be performed immediately. If the extract will be stored longer than two days, it should be transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no further cleanup, proceed with GC/MS analysis (Section 12). If the sample requires further cleanup, proceed to Section 11.

10.17Determine the original sample volume by refilling the sample bottle to the mark and transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5 mL.

11. Cleanup and Separation

11.1Cleanup procedures may not be necessary for a relatively clean sample matrix. If particular circumstances demand the use of a cleanup procedure, the analyst may use either procedure below or any other appropriate procedure.1,5-7 However, the analyst first must demonstrate that the requirements of Section 8.2 can be met using the method as revised to incorporate the cleanup procedure. Two cleanup column options are offered to the analyst in this section. The alumina column should be used first to overcome interferences. If background problems are still encountered, the silica gel column may be helpful.

11.2Alumina column cleanup for 2,3,7,8-TCDD:

11.2.1Fill a 300 mm long x 10 mm ID chromatographic column with activated alumina to the 150 mm level. Tap the column gently to settle the alumina and add 10 mm of anhydrous sodium sulfate to the top.

11.2.2Preelute the column with 50 mL of hexane. Adjust the elution rate to 1 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 1.0-mL sample extract onto the column using two 2-mL portions of hexane to complete the transfer.

11.2.3Just prior to exposure of the sodium sulfate layer to the air, add 50 mL of 3% methylene chloride/95% hexane (V/V) and continue the elution of the column. Discard the eluate.

11.2.4Next, elute the column with 50 mL of 20% methylene chloride/80% hexane (V/V) into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction to 1.0 mL as in Section 10.16 and analyze by GC/MS (Section 12).

11.3Silica gel column cleanup for 2,3,7,8-TCDD:

11.3.1Fill a 400 mm long x 11 mm ID chromatmgraphic column with silica gel to the 300 mm level. Tap the column gently to settle the silica gel and add 10 mm of anhydrous sodium sulfate to the top.

11.3.2Preelute the column with 50 mL of 20% benzene/80% hexane (V/V). Adjust the elution rate to 1 mL/min. Discard the eluate and just prior to exposure of the sodium sulfate layer to the air, quantitatively transfer the 1.0-mL sample extract onto the column using two 2-mL portions of 20% benzene/80% hexane to complete the transfer.

11.3.3Just prior to exposure of the sodium sulfate layer to the air, add 40 mL of 20% benzene/80% hexane to the column. Collect the eluate in a clean 500-mL K-D flask equipped with a 10-mL concentrator tube. Concentrate the collected fraction to 1.0 mL as in Section 10.16 and analyze by GC/MS.

12. GC/MS Analysis

12.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. Other capillary columns or chromatographic conditions may be used if the requirements of Sections 5.5.2 and 8.2 are met.

12.2Analyze standards and samples with the mass spectrometer operating in the selected ion monitoring (SIM) mode using a dwell time to give at least seven points per peak. For LRMS, use masses at m/z 320, 322, and 257 for 2,3,7,8-TCDD and either m/z 328 for 37Cl4 2,3,7,8-TCDD or m/z 332 for 13C12 2,3,7,8-TCDD. For HRMS, use masses at m/z 319.8965 and 321.8936 for 2,3,7,8-TCDD and either m/z 327.8847 for 37Cl4 2,3,7,8-TCDD or m/z 331.9367 for 13C12 2,3,7,8-TCDD.

12.3If lower detection limits are required, the extract may be carefully evaporated to dryness under a gentle stream of nitrogen with the concentrator tube in a water bath at about 40 °C. Conduct this operation immediately before GC/MS analysis. Redissolve the extract in the desired final volume of ortho-xylene or tetradecane.

12.4Calibrate the system daily as described in Section 7.

12.5Inject 2 to 5 μL of the sample extract into the gas chromatograph. The volume of calibration standard injected must be measured, or be the same as all sample injection volumes.

12.6The presence of 2,3,7,8-TCDD is qualitatively confirmed if all of the following criteria are achieved:

12.6.1The gas chromatographic column must resolve 2,3,7,8-TCDD from the other 21 TCDD isomers.

12.6.2The masses for native 2,3,7,8-TCDD (LRMS-m/z 320, 322, and 257 and HRMS-m/z 320 and 322) and labeled 2,3,7,8-TCDD (m/z 328 or 332) must exhibit a simultaneous maximum at a retention time that matches that of native 2,3,7,8-TCDD in the calibration standard, with the performance specifications of the analytical system.

12.6.3The chlorine isotope ratio at m/z 320 and m/z 322 must agree to within±10% of that in the calibration standard.

12.6.4The signal of all peaks must be greater than 2.5 times the noise level.

12.7For quantitation, measure the response of the m/z 320 peak for 2,3,7,8-TCDD and the m/z 332 peak for 13C12 2,3,7,8-TCDD or the m/z 328 peak for 37Cl4 2,3,7,8-TCDD.

12.8Co-eluting impurities are suspected if all criteria are achieved except those in Section 12.6.3. In this case, another SIM analysis using masses at m/z 257, 259, 320 and either m/a 328 or m/z 322 can be performed. The masses at m/z 257 and m/z 259 are indicative of the loss of one chlorine and one carbonyl group from 2,3,7,8-TCDD. If masses m/z 257 and m/z 259 give a chlorine isotope ratio that agrees to within ±10% of the same cluster in the calibration standards, then the presence of TCDD can be confirmed. Co-eluting DDD, DDE, and PCB residues can be confirmed, but will require another injection using the appropriate SIM masses or full repetitive mass scans. If the response for 37Cl4 2,3,7,8-TCDD at m/z 328 is too large, PCB contamination is suspected and can be confirmed by examining the response at both m/z 326 and m/z 328. The 37Cl4 2,3,7,8-TCDD internal standard gives negligible response at m/z 326. These pesticide residues can be removed using the alumina column cleanup procedure.

12.9If broad background interference restricts the sensitivity of the GC/MS analysis, the analyst should employ additional cleanup procedures and reanalyze by GC/MS.

12.10In those circumstances where these procedures do not yield a definitive conclusion, the use of high resolution mass spectrometry is suggested.5

13. Calculations

13.1Calculate the concentration of 2,3,7,8-TCDD in the sample using the response factor (RF) determined in Section 7.1.2 and Equation 2.

EC15NO91.123Equation 2where:As=SIM response for 2,3,7,8-TCDD at m/z 320.Ais=SIM response for the internal standard at m/z 328 or 332.Is=Amount of internal standard added to each extract (μg).Vo=Volume of water extracted (L).

13.2For each sample, calculate the percent recovery of the internal standard by comparing the area of the m/z peak measured in the sample to the area of the same peak in the calibration standard. If the recovery is below 50%, the analyst should review all aspects of his analytical technique.

13.3Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentration listed in Table 1 was obtained using reagent water.14 The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method was tested by 11 laboratories using reagent water, drinking water, surface water, and three industrial waste-waters spiked at six concentrations over -the range 0.02 to 0.20 μg/L.15 Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 3.

4. ASTM Annual Book of Standards, Part 31, D3694-78. “Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents,” American Society for Testing and Materials, Philadelphia.

Table 1—Chromatographic Conditions and Method Detection LimitParameterRetention time (min)Method detection limit (μg/L)2,3,7,8-TCDD13.10.002Column conditions: SP-2330 coated on a 60 m long × 0.25 mm ID glass column with hydrogen carrier gas at 40 cm/sec linear velocity, splitless injection using tetradecane. Column temperature held isothermal at 200°C for 1 min, then programmed at 8°C/min to 250 °C and held. Use of helium carrier gas will approximately double the retention time.Table 2—QC Acceptance Criteria—Method 613ParameterTest conc. (μg/L)Limit for s (μg/L)Range for X (μg/L)Range for P, Ps (%)2,3,7,8-TCDD0.1000.02760.0523-0.122645-129s=Standard deviation of four recovery measurements, in μg/L (Section 8.2.4).X=Average recovery for four recovery measurements, in μg/L (Section 8.2.4).P, Ps=Percent recovery measured (Section 8.3.2, Section 8.4.2).Note: These criteria are based directly upon the method performance data in Table 3. Where necessary, the limits for recovery have been broadened to assure applicability of the limits to concentrations below those used to develop Table 3.Table 3—Method Accuracy and Precision as Functions of Concentration—Method 613ParameterAccuracy, as recovery, X ′ (μg/L)Single analyst, precision, sr ′ (μ/L)Overall precision, S ′ (μ/g/L)2,3,7,8-TCDD0.86C+0.001450.13X+0.001290.19X+0.00028X′=Expected recovery for one or more measurements. of a sample containing a concentration of C, in μg/L.sr′=Expected single analyst standard deviation of measurements at an average concentration found of X, in μg/L.S′=Expected interlaboratory standard deviation of measurements at an average concentration found of X, in μg/L.C=True value for the concentration, in μg/L.X=Average recovery found for measurements of samples containing a concentration of C, in μg/L.Method 624—PurgeablesPt. 136, App. A, Meth. 6241. Scope and Application

1.1This method covers the determination of a number of purgeable organics. The following parameters may be determined by this method:

1.2The method may be extended to screen samples for acrolein (STORET No. 34210, CAS No. 107-02-8) and acrylonitrile (STORET No. 34215, CAS No. 107-13-1), however, the preferred method for these two compounds in Method 603.

1.3This is a purge and trap gas chro-ma-to-graphic/-mass spectrometer (GC/MS) method applicable to the determination of the compounds listed above in municipal and industrial discharges as provided under 40 CFR 136.1.

1.4The method detection limit (MDL, defined in Section 14.1)1 for each parameter is listed in Table 1. The MDL for a specific wastewater may differ from those listed, depending upon the nature of interferences in the sample matrix.

1.5Any modification to this method, beyond those expressly permitted, shall be considered as a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5. Depending upon the nature of the modification and the extent of intended use, the applicant may be required to demonstrate that the modifications will produce equivalent results when applied to relevant wastewaters.

1.6This method is restricted to use by or under the supervision of analysts experienced in the operation of a purge and trap system and a gas chromatograph/mass spectrometer and in the interpretation of mass spectra. Each analyst must demonstrate the ability to generate acceptable results with this method using the procedure described in Section 8.2.

2. Summary of Method

2.1An inert gas is bubbled through a 5-mL water sample contained in a specially-designed purging chamber at ambient temperature. The purgeables are efficiently transferred from the aqueous phase to the vapor phase. The vapor is swept through a sorbent trap where the purgeables are trapped. After purging is completed, the trap is heated and backflushed with the inert gas to desorb the purgeables onto a gas chromatographic column. The gas chromatograph is temperature programmed to separate the purgeables which are then detected with a mass spectrometer.2,3

3. Interferences

3.1Impurities in the purge gas, organic compounds outgassing from the plumbing ahead of the trap, and solvent vapors in the laboratory account for the majority of contamination problems. The analytical system must be demonstated to be free from contamination under the conditions of the analysis by running laboratory reagent blanks as described in Section 8.1.3. The use of non-Teflon plastic tubing, non-Teflon thread sealants, or flow controllers with rubber components in the purge and trap system should be avoided.

3.2Samples can be contaminated by diffusion of volatile organics (particularly fluorocarbons and methylene chloride) through the septum seal into the sample during shipment and storage. A field reagent blank prepared from reagent water and carried through the sampling and handling protocol can serve as a check on such contamination.

3.3Contamination by carry-over can occur -whenever high level and low level samples are sequentially analyzed. To reduce carry-over, the purging device and sample syringe must be rinsed with reagent water between sample analyses. Whenever an unusually concentrated sample is encountered, it should be followed by an analysis of reagent water to check for cross contamination. For samples containing large amounts of water-soluble materials, suspended solids, high boiling compounds or high pureeable levels, it may be necessary to wash the purging device with a detergent solution, rinse it with distilled water, and then dry it in a 105 °C oven between analyses. The trap and other parts of the system are also subject to contamination; therefore, frequent bakeout and purging of the entire system may be required.

4. Safety

4.1The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever means available. The laboratory is responsible for maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this methmd. A reference file of material data handling sheets should also be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available and have been identified4-6 for the information of the analyst.

4.2.The following parameters covered by this method have been tentatively classified as known or suspected, human or mammalian carcinogens: benzene, carbon tetrachloride, chloroform, 1,4-dichlorobenzene, and vinyl chloride. Primary standards of these toxic compounds should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be worn when the analyst handles high concentrations of these toxic compounds.

5. Apparatus and Materials

5.1Sampling equipment, for discrete sampling.

5.1.1Vial—25-mL capacity or larger, equipped -with a screw cap with a hole in the center (Pierce #13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at 105 °C before use.

5.2Purge and trap system—The purge and trap system consists of three separate pieces of equipment: A purging device, trap, and desorber. Several complete systems are now commercially available.

5.2.1The purging device must be designed to accept 5-mL samples with a water column at least 3 cm deep. The gaseous head space between the water column and the trap must have a total volume of less than 15 mL. The purge gas must pass though the water column as finely divided bubbles with a diameter of less than 3 mm at the origin. The purge gas must be introduced no more than 5 mm from the base of the water column. The purging device illustrated in Figure 1 meets these design criteria.

5.2.2The trap must be at least 25 cm long and have an inside diameter of at least 0.105 in. The trap must be packed to contain the following minimum lengths of adsorbents: 1.0 cm of methyl silicone coated packing (Section 6.3.2), 15 cm of 2,6-dyphenylene oxide polymer (Section 6.3.1), and 8 cm of silica gel (Section 6.3.3). The minimum specifications for the trap are illustrated in Figure 2.

5.2.3The desorber should be capable of rapidly heating the trap to 180 °C. The polymer section of the trap should not be heated higher than 180 °C and the remaining sections should not exceed 200 °C. The desorber illustrated in Figure 2 meets these design criteria.

5.2.4The purge and trap system may be assembled as a separate unit or be coupled to a gas chromatograph as illustrated in Figures 3 and 4.

5.3GC/MS system:

5.3.1Gas chromatograph—An analytical system complete with a temperature programmable gas chromatograph suitable for on-column injection and all required accessories including syringes, analytical columns, and gases.

5.3.2Column—6 ft long x 0.1 in ID stainless steel or glass, packed with 1% SP-1000 on Carbopack B (60/80 mesh) or equivalent. This column was used to develop the method performance statements in Section 14. Guidelines for the use of alternate column packings are provided in Section 11.1.

5.3.3Mass spectrometer—Capable of scanning from 20 to 260 amu every 7 s or less, utilizing 70 V (nominal) electron energy in the electron impact ionization mode, and producing a mass spectrum which meets all the criteria in Table 2 when 50 ng of 4-bromo-flu-o-ro-ben-zene (BFB) is injected through the GC inlet.

5.3.4GC/MS interface—Any GC to MS interface that gives acceptable calibration points at 50 ng or less per injection for each of the parameters of interest and achieves all acceptable performance criteria (Section 10) may be used. GC to MS interfaces constructed of all glass or glass-lined materials are recommended. Glass can be deactivated by silanizing with dichlorodimethylsilane.

5.3.5Data system—A computer system must be interfaced to the mass spectrometer that allows the continuous acquisition and storage on machine-readable media of all mass spectra obtained throughout the duration of the chromatographic program. The computer must have software that allows searching any GC/MS data file for specific m/z (masses) and plotting such m/z abundances versus time or scan number. This type of plot is defined as an Extracted Ion Current Profile (EICP). Software must also be available that allows integrating the abundance in any EICP between specified time or scan number limits.

6.1Reagent water—Reagent water is defined as a water in which an interferent is not observed at the MDL of the parameters of interest.

6.1.1Reagent water can be generated by passing tap water through a carbon filter bed containing about 1 lb of activated carbon (Filtrasorb-300, Calgon Corp., or equivalent).

6.1.2A water purification system (Millipore Super-Q or equivalent) may be used to generate reagent water.

6.1.3Reagent water may also be prepared by boiling water for 15 min. Subsequently, while maintaining the temperature at 90 °C, bubble a contaminant-free inert gas through the water for 1 h. While still hot, transfer the water to a narrow mouth screw-cap bottle and seal with a Teflon-lined septum and cap.

6.5Stock standard solutions—Stock stand-ard solutions may be prepared from pure -standard materials or purchased as certi-fied solutions. Prepare stock standard solutions in methanol using assayed liquids or gases as appropriate. Because of the toxicity of some of the compounds, primary dilutions of these materials should be prepared in a hood. A NIOSH/MESA approved toxic gas respirator should be used when the analyst handles high concentrations of such materials.

6.5.1Place about 9.8 mL of methanol into a 10-mL ground glass stoppered volumetric flask. Allow the flask to stand, unstoppered, for about 10 min or until all alcohol wetted surfaces have dried. Weigh the flask to the nearest 0.1 mg.

6.5.2Add the assayed reference material:

6.5.2.1Liquids—Using a 100-μL syringe, immediately add two or more drops of assayed reference material to the flask, then reweigh. Be sure that the drops fall directly into the alcohol without contacting the neck of the flask.

6.5.2.2Gases—To prepare standards for any of the four halocarbons that boil below 30 °C (bromomethane, chloroethane, chloromethane, and vinyl chloride), fill a 5-mL valved gas-tight syringe with the reference standard to the 5.0-mL mark. Lower the needle to 5 mm above the methanol meniscus. Slowly introduce the reference standard above the surface of the liquid (the heavy gas will rapidly dissolve in the methanol).

6.5.3Reweigh, dilute to volume, stopper, then mix by inverting the flask several times. Calculate the concentration in μg/μL from the net gain in weight. When compound purity is assayed to be 96% or greater, the weight may be used without correction to calculate the concentration of the stock standard. Commercially prepared stock standards may be used at any concentration if they are certified by the manufacturer or by an independent source.

6.5.4Transfer the stock standard solution into a Teflon-sealed screw-cap bottle. Store, with minimal headspace, at −10 to −20 °C and protect from light.

6.5.5Prepare fresh standards weekly for the four gases and 2-chloroethylvinyl ether. All other standards must be replaced after one month, or sooner if comparison with check standards indicates a problem.

6.6Secondary dilution standards—Using stock solutions, prepare secondary dilution standards in methanol that contain the compounds of interest, either singly or mixed together. The secondary dilution standards should be prepared at concentrations such that the aqueous calibration standards prepared in Section 7.3 will bracket the working range of the analytical system. Secondary dilution standards should be stored with minimal headspace and should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.

6.7Surrogate standard spiking solution—Select a minimum of three surrogate compounds from Table 3. Prepare stock standard solutions for each surrogate standard in methanol as described in Section 6.5. Prepare a surrogate standard spiking solution from these stock standards at a concentration of 15 μg/mL in water. Store the solutions at 4 °C in Teflon-sealed glass containers with a minimum of headspace. The solutions should be checked frequently for stability. The addition of 10 μL of this solution of 5 mL of sample or standard is equivalent to a concentration of 30 μg/L of each surrogate standard.

6.8BFB Standard—Prepare a 25 μg/mL solution of BFB in methanol.

6.9Quality control check sample concentrate—See Section 8.2.1.

7. Calibration

7.1Assemble a purge and trap system that meets the specifications in Section 5.2. Condition the trap overnight at 180 °C by backflushing with an inert gas flow of at least 20 mL/min. Condition the trap for 10 min once daily prior to use.

7.2Connect the purge and trap system to a gas chromatograph. The gas chromatograph must be operated using temperature and flow rate conditions equivalent to those given in Table 1.

7.3Internal standard calibration procedure—To use this approach, the analyst must select three or more internal standards that are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate that the measurement of the internal standard is not affected by method or matrix interferences. Some recommended internal standards are listed in Table 3.

7.3.1Prepare calibration standards at a minimum of three concentration levels for each parameter by carefully adding 20.0 μL of one or more secondary dilution standards to 50, 250, or 500 mL of reagent water. A 25-μL syringe with a 0.006 in. ID needle should be used for this operation. One of the calibration standards should be at a concentration near, but above, the MDL (Table 1) and the other concentrations should correspond to the expected range of concentrations found in real samples or should define the working range of the GC/MS system. These aqueous standards can be stored up to 24 h, if held in sealed vials with zero headspace as described in Section 9.2. If not so stored, they must be discarded after 1 h.

7.3.2Prepare a spiking solution containing each of the internal standards using the procedures described in Sections 6.5 and 6.6. It is recommended that the secondary dilution standard be prepared at a concentration of 15 μg/mL of each internal standard compound. The addition of 10 μL of this standard to 5.0 mL of sample or calibration standard would be equivalent to 30 μg/L.

7.3.3Analyze each calibration standard according to Section 11, adding 10 μL of internal standard spiking solution directly to the syringe (Section 11.4). Tabulate the area response of the characteristic m/z against concentration for each compound and internal standard, and calculate response factors (RF) for each compound using Equation 1.

EC15NO91.124Equation 1where:As=Area of the characteristic m/z for the parameter to be measured.Ais=Area of the characteristic m/z for the inernal standard.Cis=Concentration of the internal standard.Cs=Concentration of the parameter to be measured.If the RF value over the working range is a constant (<35% RSD), the RF can be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can be used to plot a calibration curve of response ratios, As/Ais, vs. RF.

7.4The working calibration curve or RF must be verified on each working day by the measurement of a QC check sample.

7.4.1Prepare the QC check sample as described in Section 8.2.2.

7.4.2Analyze the QC check sample according to the method beginning in Section 10.

7.4.3For each parameter, compare the response (Q) with the corresponding calibration acceptance criteria found in Table 5. If the responses for all parameters of interest fall within the designated ranges, analysis of actual samples can begin. If any individual Q falls outside the range, proceed according to Section 7.4.4.

Note:

The large number of parameters in Table 5 present a substantial probability that one or more will not meet the calibration acceptance criteria when all parameters are analyzed.

7.4.4Repeat the test only for those parameters that failed to meet the calibration acceptance criteria. If the response for a parameter does not fall within the range in this second test, a new calibration curve or RF must be prepared for that parameter according to Section 7.3.

8. Quality Control

8.1Each laboratory that uses this method is required to operate a formal quality control program. The minimum requirements of this program consist of an initial demonstration of laboratory capability and an ongoing analysis of spiked samples to evaluate and document data quality. The laboratory must maintain records to document the quality of data that is generated. Ongoing data quality checks are compared with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. When results of sample spikes indicate atypical method performance, a quality control check standard must be analyzed to confirm that the measurements were performed in an in-control mode of operation.

8.1.1The analyst must make an initial, one-time, demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2In recognition of advances that are occurring in chromatography, the analyst is permitted certain options (detailed in Section 11.1) to improve the separations or lower the cost of measurements. Each time such a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2.

8.1.3Each day, the analyst must analyze a reagent water blank to demonstrate that interferences from the analytical system are under control.

8.1.4The laboratory must, on an ongoing basis, spike and analyze a minimum of 5% of all samples to monitor and evaluate laboratory data quality. This procedure is described in Section 8.3.

8.1.5The laboratory must, on an ongoing basis, demonstrate through the analyses of quality control check standards that the operation of the measurement system is in control. This procedure is described in Section 8.4. The frequency of the check standard analyses is equivalent to 5% of all samples analyzed but may be reduced if spike recoveries from samples (Section 8.3) meet all specified quality control criteria.

8.1.6The laboratory must spike all samples with surrogate standards to monitor continuing laboratory performance. This procedure is described in Section 8.5.

8.1.7The laboratory must maintain performance records to document the quality of data that is generated. This procedure is described in Section 8.6.

8.2To establish the ability to generate acceptable accuracy and precision, the analyst must perform the following operations.

8.2.1A quality control (QC) check sample concentrate is required containing each parameter of interest at a concentration of 10 μg/mL in methanol. The QC check sample concentrate must be obtained from the U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory in Cincinnati, Ohio, if available. If not available from that source, the QC check sample concentrate must be obtained from another external source. If not available from either source above, the QC check sample concentrate must be prepared by the laboratory using stock standards prepared independently from those used for calibration.

8.2.3Analyze four 5-mL aliquots of the well-mixed QC check sample according to the method beginning in Section 10.

8.2.4Calculate the average recovery (X) in μg/L, and the standard deviation of the recovery (s) in μg/L, for each parameter of interest using the four results.

8.2.5For each parameter compare s and X with the corresponding acceptance criteria for precision and accuracy, respectively, found in Table 5. If s and X for all parameters of interest meet the acceptance criteria, the system performance is acceptable and analysis of actual samples can begin. If any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, the system performance is unacceptable for that parameter.

Note:

The large number of parameters in Table 5 present a substantial probability that one or more will fail at least one of the acceptance criteria when all parameters are analyzed.

8.2.6When one or more of the parameters tested fail at least one of the acceptance criteria, the analyst must proceed according to Section 8.2.6.1 or 8.2.6.2.

8.2.6.1Locate and correct the source of the problem and repeat the test for all parameters of interest beginning with Section 8.2.3.

8.2.6.2Beginning with Section 8.2.3, repeat the test only for those parameters that failed to meet criteria. Repeated failure, however, will confirm a general problem with the measurement system. If this occurs, locate and correct the source of the problem and repeat the test for all compounds of interest beginning with Section 8.2.3.

8.3The laboratory must, on an ongoing basis, spike at least 5% of the samples from each sample site being monitored to assess accuracy. For laboratories analyzing 1 to 20 samples per month, at least one spiked sample per month is required.

8.3.1The concentration of the spike in the sample should be determined as follows:

8.3.1.1If, as in compliance monitoring, the concentration of a specific parameter in the sample is being checked against a regulatory concentration limit, the spike should be at that limit or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.1.2If the concentration of a specific parameter in the sample is not being checked against a limit specific to that parameter, the spike should be at 20 μg/L or 1 to 5 times higher than the background concentration determined in Section 8.3.2, whichever concentration would be larger.

8.3.2Analyze one 5-mL sample aliquot to determine the background concentration (B) of each parameter. If necessary, prepare a new QC check sample concentrate (Section 8.2.1) appropriate for the background concentrations in the sample. Spike a second 5-mL sample aliquot with 10 μL of the QC check sample concentrate and analyze it to determine the concentration after spiking (A) of each parameter. Calculate each percent recovery (P) as 100(A-B)%/T, where T is the known true value of the spike.

8.3.3Compare the percent recovery (P) for each parameter with the corresponding QC acceptance criteria found in Table 5. These acceptance criteria wer calculated to include an allowance for error in measurement of both the background and spike concentrations, assuming a spike to background ratio of 5:1. This error will be accounted for to the extent that the analyst's spike to background ratio approaches 5:1.7 If spiking was performed at a concentration lower than 20 μg/L, the analyst must use either the QC acceptance criteria in Table 5, or optional QC acceptance criteria calculated for the specific spike concentration. To calculate optional acceptance criteria for the recoveryof a parameter: (1) Calculate accuracy (X′) using the equation in Table 6, substituting the spike concentration (T) for C; (2) calculate overall precision (S′) using the equation in Table 6, substituting X′ for X; (3) calculate the range for recovery at the spike concentration as (100 X′/T) (±2.44(100 S′/T)%.7

8.3.4If any individual P falls outside the designated range for recovery, that parameter has failed the acceptance criteria. A check standard containing each parameter that failed the criteria must be analyzed as described in Section 8.4.

8.4If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC check standard containing each parameter that failed must be prepared and analyzed.

Note:

The frequency for the required anlaysis of a QC check standard will depend upon the number of parameters being simultaneously tested, the complexity of the sample matrix, and the performance of the laboratory. If the entire list of parameters in Table 5 must be measured in the sample in Section 8.3, the probability that the analysis of a QC check standard will be required is high. In this case the QC check standard should be routinely analyzed with the spiked sample.

8.4.1Prepare the QC check standard by adding 10 μL of QC check sample concentrate (Section 8.2.1 or 8.3.2) to 5 mL of reagent water. The QC check standard needs only to contain the parameters that failed criteria in the test in Section 8.3.

8.4.2Analyze the QC check standard to determine the concentration measured (A) of each parameter. Calculate each percent recovery (PS) as 100 (A/T)%, where T is the true value of the standard concentration.

8.4.3Compare the percent recovery (PS) for each parameter with the corresponding QC acceptance criteria found in Table 5. Only parameters that failed the test in Section 8.3 need to be compared with these criteria. If the recovery of any such parameter falls outside the designated range, the laboratory performance for that parameter is judged to be out of control, and the problem must be immediately identified and corrected. The analytical result for that parameter in the unspiked sample is suspect and may not be reported for regulatory compliance purposes.

8.5As a quality control check, the laboratory must spike all samples with the surrogate standard spiking solutions as described in Section 11.4, and calculate the percent recovery of each surrogate compound.

8.6As part of the QC program for the laboratory, method accuracy for wastewater samples must be assessed and records must be maintained. After the analysis of five spiked wastewater samples as in Section 8.3, calculate the average percent recovery (P) and the standard deviation of the percent recovery (sp). Express the accuracy assessment as a percent recovery interval from P—2sp to P + 2sp. If P=90% and sp=10%, for example, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each parameter a regular basis (e.g. after each five to ten new accuracy measurements).

8.7It is recommended that the laboratory adopt additional quality assurance practices for use with this method. The specific practices that are most productive depend upon the needs of the laboratory and the nature of the samples. Field duplicates may be analyzed to assess the precision of the environmental measurements. Whenever possible, the laboratory should analyze standard reference materials and participate in relevant performance evaluation studies.

9. Sample Collection, Preservation, and Handling

9.1All samples must be iced or refrigerated from the time of collection until analysis. If the sample contains residual chlorine, add sodium thiosulfate preservative (10 mg/40 mL is sufficient for up to 5 ppm Cl2) to the empty sample bottle just prior to shipping to the sampling site. EPA Methods 330.4 and 330.5 may be used for measurement of residual chlorine.8 Field test kits are available for this purpose.

9.2Grab samples must be collected in glass containers having a total volume of at least 25 mL. Fill the sample bottle just to overflowing in such a manner that no air bubbles pass through the sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it. If preservative has been added, shake vigorously for 1 min. Maintain the hermetic seal on the sample bottle until time of analysis.

9.3Experimental evidence indicates that some aromatic compounds, notably benzene, toluene, and ethyl benzene are susceptible to rapid biological degradation under certain environmental conditions.3 Refrigeration alone may not be adequate to preserve these compounds in wastewaters for more than seven days. For this reason, a separate sample should be collected, acidified, and analyzed when these aromatics are to be determined. Collect about 500 mL of sample in a clean container. Adjust the pH of the sample to about 2 by adding 1+1 HCl while stirring vigorously, Check pH with narrow range (1.4 to 2.8) pH paper. Fill a sample container as described in Section 9.2.

9.4All samples must be analyzed within 14 days of collection.3

10. Daily GC/MS Performance Tests

10.1At the beginning of each day that analyses are to be performed, the GC/MS system must be checked to see if acceptable performance criteria are achieved for BFB.9 The performance test must be passed before any samples, blanks, or standards are analyzed, unless the instrument has met the DFTPP test described in Method 625 earlier in the day.10

10.3At the beginning of each day, inject 2 μL of BFB solution directly on the column. Alternatively, add 2 μL of BFB solution to 5.0 mL of reagent water or standard solution and analyze the solution according to section 11. Obtain a background-corrected mass spectrum of BFB and confirm that all the key m/z criteria in Table 2 are achieved. If all the criteria are not achieved, the analyst must retune the mass spectrometer and repeat the test until all criteria are achieved.

11. Sample Purging and Gas Chromatography

11.1Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in this table are retention times and MDL that can be achieved under these conditions. An example of the separations achieved by this column is shown in Figure 5. Other packed columns or chromatographic conditions may be used if the requirements of Section 8.2 are met.

11.2After achieving the key m/z abundance criteria in Section 10, calibrate the system daiy as described in Section 7.

11.3Adjust the purge gas (helium) flow rate to 40 mL/min. Attach the trap inlet to the purging device, and set the purge and trap system to purge (Figure 3). Open the syringe valve located on the purging device sample introduction needle.

11.4Allow the sample to come to ambient temperature prior to introducing it into the syringe. Remove the plunger from a 5-mL syringe and attach a closed syringe valve. Open the sample bottle (or standard) and carefully pour the sample into the syringe barrel to just short of overflowing. Replace the syringe plunger and compress the sample. Open the syringe valve and vent any residual air while adjusting the sample volume to 5.0 mL. Since this process of taking an aliquot destroys the validity of the sample for future analysis, the analyst should fill a second syringe at this time to protect against possible loss of data. Add 10.0 μL of the surrogate spiking solution (Section 6.7) and 10.0 μL of the internal standard spiking solution (Section 7.3.2) through the valve bore, then close the valve. The surrogate and internal standards may be mixed and added as a single spiking solution.

11.5Attach the syringe-syringe valve assembly to the syringe valve on the purging device. Open the syringe valves and inject the sample into the purging chamber.

11.6Close both valves and purge the sample for 11.0±0.1 min at ambient temperature.

11.7After the 11-min purge time, attach the trap to the chromatograph, adjust the purge and trap system to the desorb mode (Figure 4), and begin to temperature program the gas chromatograph. Introduce the trapped materials to the GC column by rapidly heating the trap to 180 °C while backflushing the trap with an inert gas between 20 and 60 mL/min for 4 min. If rapid heating of the trap cannot be achieved, the GC cloumn must be used as a secondary trap by cooling it to 30 °C (subambient temperature, if problems persist) instead of the initial program temperature of 45 °C.

11.8While the trap is being desorbed into the gas chromatograph, empty the purging chamber using the sample introduction syringe. Wash the chamber with two 5-mL flushes of reagent water.

11.9After desorbing the sample for 4 min, recondition the trap by returning the purge and trap system to the purge mode. Wait 15 s then close the syringe valve on the purging device to begin gas flow through the trap. The trap temperature should be maintained at -180 °C. After approximately 7 min, turn off the trap heater and open the syringe valve to stop the gas flow through the trap. When the trap is cool, the next sample can be analyzed.

11.10If the response for any m/z exceeds the working range of the system, prepare a dilution of the sample with reagent water from the aliquot in the second syringe and reanalyze.

12. Qualitative Identification

12.1Obtain EICPs for the primary m/z (Table 4) and at least two secondary masses for each parameter of interest. The following criteria must be met to make a qualitative identification:

12.1.1The characteristic masses of each parameter of interest must maximize in the same or within one scan of each other.

12.1.2The retention time must fall within ±30 s of the retention time of the authentic compound.

12.1.3The relative peak heights of the three characteristic masses in the EICPs must fall within ±20% of the relative intensities of these masses in a reference mass spectrum. The reference mass spectrum can be obtained from a standard analyzed in the GC/MS system or from a reference library.

12.2Structural isomers that have very similar mass spectra and less than 30 s difference in retention time, can be explicitly identified only if the resolution between authentic isomers in a standard mix is acceptable. Acceptable resolution is achieved if the baseline to valley height between the isomers is less than 25% of the sum of the two peak heights. Otherwise, structural isomers are identified as isomeric pairs.

13. Calculations

13.1When a parameter has been identified, the quantitation of that parameter should be based on the integrated abundance from the EICP of the primary characteristic m/z given in Table 4. If the sample produces an interference for the primary m/z, use a secondary characteristic m/z to quantitate.

Calculate the concentration in the sample using the response factor (RF) determined in Section 7.3.3 and Equation 2.

EC15NO91.125Equation 2where:AS=Area of the characteristic m/z for the parameter or surrogate standard to be measured.Ais=Area of the characteristic m/z for the internal standard.Cis=Concentration of the internal standard.

13.2Report results in μg/L without correction for recovery data. All QC data obtained should be reported with the sample results.

14. Method Performance

14.1The method detection limit (MDL) is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the value is above zero.1 The MDL concentrations listed in Table 1 were obtained using reagent water.11 Similar results were achieved using representative wastewaters. The MDL actually achieved in a given analysis will vary depending on instrument sensitivity and matrix effects.

14.2This method was tested by 15 laboratories using reagent water, drinking water, surface water, and industrial wastewaters spiked at six concentrations over the range 5-600 μg/L.12Single operator precision, overall precision, and method accuracy were found to be directly related to the concentration of the parameter and essentially independent of the sample matrix. Linear equations to describe these relationships are presented in Table 5.

7. Provost, L.P., and Elder, R.S. “Interpretation of Percent Recovery Data,” American Laboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.2.3 is two times the value 1.22 derived in this report.)

8. “Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPD) for Chlorine, Total Residual,” Methods for Chemical Analysis of Water and Wastes, EPA-600/4-79-020, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979.